Methods of antagonizing OP-1 binding to a cell surface receptor utilizing ALK polypeptides

ABSTRACT

Disclosed are (1) nucleic acid sequences, amino acid sequences, homologies, structural features and various other data characterizing a morphogen cell surface receptors particularey OP-1-binding cell surface receptors; (2) methods for producing receptor proteins, including fragments thereof, using recombinant DNA technology; (3) methods for identifying novel morphogen receptors and their encoding DNAs; (4) methods and compositions for identifying compounds capable of modulating endogenous morphogen receptor levels; and (5) methods and compositions for identifying morphogen receptor binding analogs useful in the design of morphogen agonists and antagonists for therapeutic, diagnostic and experimental uses.

This application is filed under 35 U.S.C. § 371 as a national stageapplication of PCT/US95/05467, filed Apr. 28, 1995, and acontinuation-in-part of U.S. application Ser. No. 08/236,428, filed Apr.29, 1994, now abandoned.

FIELD OF THE INVENTION

This invention relates generally to the field of tissue morphogenesisand more particularly to morphogenic protein-specific cell surfacereceptors.

BACKGROUND OF THE INVENTION

Cell differentiation is the central characteristic of tissuemorphogenesis which initiates during embryogenesis, and continues tovarious degrees throughout the life of an organism in adult tissuerepair and regeneration mechanisms. The degree of morphogenesis in adulttissue varies among different tissues and is related, among otherthings, to the degree of cell turnover in a given tissue.

The cellular and molecular events which govern the stimulus fordifferentiation of cells is an area of intensive research. In themedical and veterinary fields, it is anticipated that the discovery ofthe factor or factors which control cell differentiation and tissuemorphogenesis will advance significantly medicine's ability to repairand regenerate diseased or damaged mammalian tissues and organs.Particularly useful areas for human and veterinary therapeutics includereconstructive surgery and in the treatment of tissue degenerativediseases including arthritis, emphysema, osteoporosis, cardiomyopathy,cirrhosis, degenerative nerve diseases, inflammatory diseases, andcancer, and in the regeneration of tissues, organs and limbs. (In thisand related applications, the terms "morphogenetic" and "morphogenic"are used interchangeably.) A number of different factors have beenisolated in recent years which appear to play a role in celldifferentiation. Recently, a distinct subfamily of the "superfamily" ofstructurally related proteins referred to in the art as the"transforming growth factor-b (TGF-β) superfamily of proteins" have beenidentified as true tissue morphogens.

The members of this distinct "subfamily" of true tissue morphogenicproteins share substantial amino acid sequence homology within theirmorphogenetically active C-terminal domains (at least 50% identity inthe C-terminal 102 amino acid sequence), including a conserved six orseven cysteine skeleton, and share the in vivo activity of inducingtissue-specific morphogenesis in a variety of organs and tissues. Theproteins apparently contact and interact with progenitor cells e.g., bybinding suitable cell surface molecules, predisposing or otherwisestimulating the cells to proliferate and differentiate in amorphogenetically permissive environment. These morphogenic proteins arecapable of inducing the developmental cascade of cellular and molecularevents that culminate in the formation of new organ-specific tissue,including any vascularization, connective tissue formation, and nerveinnervation as required by the naturally occurring tissue. The proteinshave been shown to induce morphogenesis of both bone cartilage and bone,as well as periodontal tissues, dentin, liver, and neural tissue,including retinal tissue.

The true tissue morphogenic proteins identified to date include proteinsoriginally identified as bone inductive proteins. These include OP-1,(osteogenic protein-1, also referred to in related applications as"OP1"), its Drosophila homolog, 60A, with which it shares 69% identityin the C-terminal "seven cysteine" domain, and the related proteins OP-2(also referred to in related applications as "OP2") and OP-3, both ofwhich share approximately 70-75% identity with OP-1 in the C-terminalseven cysteine domain, as well as BMP5, BMP6 and its murine homolog,Vgr-1, all of which share greater than 85% identity with OP-1 in theC-terminal seven cysteine domain, and the BMP6 Xenopus homolog, Vgl,which shares approximately 57% identity with OP-1in the C-terminal sevencysteine domain. Other bone inductive proteins include the CBMP2proteins (also referred to in the art as BMP2 and BMP4) and theirDrosophila homolog, DPP. Another tissue morphogenic protein is GDF-1(from mouse). See, for example, PCT documents US92/01968 and US92/07358,the disclosures of which are incorporated herein by reference.

As stated above, these true tissue morphogenic proteins are recognizedin the art as a distinct subfamily of proteins different from othermembers of the TGF-β superfamily in that they share a high degree ofsequence identity in the C-terminal domain and in that the true tissuemorphogenic proteins are able to induce, on their own, the full cascadeof events that result in formation of functional tissue rather thanmerely inducing formation of fibrotic (scar) tissue. Specifically,members of the family of morphogenic proteins are capable of all of thefollowing in a morphogenetically permissive environment: stimulatingcell proliferation and cell differentiation, and supporting the growthand maintenance of differentiated cells. The morphogenic proteinsapparently may act as endocrine, paracrine or autocrine factors.

The morphogenic proteins are capable of significant species "crosstalk."That is, xenogenic (foreign species) homologs of these proteins cansubstitute for one another in functional activity. For example, DPP and60A, two Drosophila proteins, can substitute for their mammalianhomologs, BMP2/4 and OP-1, respectively, and induce endochondral boneformation at a non-bony site in a standard rat bone formation assay.Similarly, BMP2 has been shown to rescue a dpp⁻ mutation in Drosophila.In their native form, however, the proteins appear to betissue-specific, each protein typically being expressed in or providedto one or only a few tissues or, alternatively, expressed only atparticular times during development. For exhample, GDF-1 appears to beexpressed primarily in neural tissue, while OP-2 appears to be expressedat relatively high levels in early (e.g., 8-day) mouse embryos. Theendogenous morphogens may be synthesized by the cells on which they act,by neighboring cells, or by cells of a distant tissue, the secretedprotein being transported to the cells to be acted on.

A particularly potent tissue morphogenic protein is OP-1. This protein,and its xenogenic homologs, are expressed in a number of tissues,primarily in tissues of urogenital origin, as well as in bone, mammaryand salivary gland tissue, reproductive tissues, and gastrointestinaltract tissue. It is also expressed in different tissues duringembryogenesis, its presence coincident with the onset of morphogenesisof that tissue.

The morphogenic protein signal transduction across a cell membraneappears to occur as a result of specific binding interaction with one ormore cell surface receptors. Recent studies on cell surface receptorbinding of various members of the TGF-β protein superfamily suggeststhat the ligands can mediate their activity by interaction with twodifferent receptors, referred to as Type I and Type II receptors to forma hetero-complex. A cell surface bound beta-glycan also may enhance thebinding interaction. The Type I and Type II receptors are bothserine/threonine kinases, and share similar structures: an intracellulardomain that consists essentially of the kinase, a short, extendedhydrophobic sequence sufficient to span the membrane one time, and anextracellular domain characterized by a high concentration of conservedcysteines.

A number of Type II receptor sequences recently have been identified.These include "TGF-βR II", a TGF-β Type II receptor (Lin et al. (1992)Cell 68:775-785); and numerous activin-binding receptors. See, forexample, Mathews et al. (1991) Cell 65:973-982 and international patentapplication WO 92/20793, published Nov. 26, 1992, disclosing the "ActRII" sequence; Attisano et al., (1992) Cell 68:97-108, disclosing the"ActR-IIB" sequence; and Legerski et al. (1992) Biochem Biophys. Res.Commun 183:672-679. A different Type II receptor shown to have affinityfor activin is Atr-II (Childs et al.(1993) PNAS 90:9475-9479.) Two TypeII receptors have been identified in C. elegans, the daf-1 gene, (Georgiet al. (1990) Cell 61:635-645), having no known ligand to date, anddaf-4, which has been shown to bind BMP4, but not activin or TGF-β(Estevez, et al. (1993) Nature 365:644-649.)

Ten Dijke et al. disclose the cloning of six different Type I cellsurface receptors from murine and human cDNA libraries. ((1993) Oncogene8:2879-2887, and Science (1994) 264:101-104. These receptors, referencedas ALK-1 to ALK-6 ("activin receptor-like kinases"), share significantsequence identities (60-79%) and several have been identified as TGF-βbinding (ALK-5) or activin binding (ALK-2, ALK-4) receptors. Xie et al.also report a Drosophila Type I receptor encoded by the sax gene(Science (1994) 263:1756-1759). The authors suggest that the proteinbinds DPP.

To date, the Type I receptors with which the morphogenic proteinsdescribed herein interact on the cell surface have not yet beenidentified, and no Type II receptor has been described as having bindingaffinity for OP-1 and its related sequences. Identification of thesecell surface molecules, with which the morphogens interact and throughwhich they may mediate their biological effect, is anticipated toenhance elucidation of the molecular mechanism of tissue morphogenesisand to enable development of morphogen receptor binding "analogs", e.g.,compounds (which may or may not be amino acid-based macromolecules)capable of mimicking the binding affinity of a morphogen for itsreceptor sufficiently to act either as a receptor binding agonist orantagonist. These "analogs" have particular utility in therapeutic,diagnostic and experimental research applications.

It is an object of this invention to provide nucleic acid molecules andamino acid sequences encoding morphogenic protein binding cell surfacereceptors, particularly OP-1-specific binding receptor sequences.Another object is to provide methods for identifying genes in a varietyof species and/or tissues, and in a variety of nucleic acid librariesencoding morphogenic protein binding receptors, particularly receptorsthat bind OP-1. Still another object is to provide means for designingbiosynthetic receptor-binding ligand analogs, particularly OP-1 analogs,and/or for identifying natural-occurring ligand analogs, includingagonists and antagonists, using the receptor molecules described herein,and analogs thereof. Another object is to provide antagonists, includingsoluble receptor constructs comprising the extracellular ligand-bindingdomain, which can modulate the availability of OP1 for receptor bindingin vivo. Another object is to provide means and compositions forcompeting with activin-receptor and BMP2/4-receptor interactions. Yetanother object is to provide means and compositions for ligand affinitypurification and for diagnostic detection and quantification of ligandsin a body fluid using OP1-specific cell surface receptors andligand-binding fragments thereof. Still another object is to providesmeans and compositions for modulating the endogenous expression orconcentration of these receptor molecules. Yet another object is toprovide ligand-receptor complexes and analog sequences thereof, as wellas antibodies capable of identifying and distinguishing the complex fromits component proteins. Still another object is to provide means andcompositions for modulating a morphogenesis in a mammal. These and otherobjects and features of the invention will be apparent from thedescription, drawings and claims which follow.

SUMMARY OF THE INVENTION

Type I and Type II cell surface receptor molecules capable of specificbinding affinity with true tissue morphogenic proteins, particularlyOP-1 related proteins, now have been identified. Accordingly, theinvention provides ligand-receptor complexes comprising at least theligand binding domain of these receptors and OP-1 or an OP-1receptor-binding analog as the ligand; means for identifying and/ordesigning useful OP-1 receptor-binding analogs and OP-1-binding-receptor analogs; and means for modulating the tissue morphogenesiscapability of a cell.

The morphogen cell surface receptors useful in this invention arereferred to in the art as Type I or Type II serine/threonine kinasereceptors. They share a conserved structure, including an extracellular,ligand-binding domain generally composed of about 100-130 amino acids(Type I receptors; up to about 196 amino acids for Type II receptors), atransmembrane domain sufficient to span a cellular membrane one time,and an intracellular (cytoplasmic) domain having serine/threonine kinaseactivity. The intact receptor is a single polypeptide chain of about500-550 amino acids and having an apparent molecular weight of about50-55 kDa.

Of particular utility in the methods and compositions of the inventionare the Type I cell surface receptors referenced herein and in theliterature as, ALK-2, ALK-3 and ALK-6, whose nucleic acids and encodedamino acid sequences are represented by the sequences in Seq. ID Nos. 3,5 and 7 respectively, and which, as demonstrated herein below, havespecific binding affinity for OP1 and OP1-related analogs. Accordingly,in one embodiment, the receptor sequences contemplated herein includeOP-1 binding analogs of the ALK-2, ALK-3 and ALK-6 proteins describedherein.

As used herein, ligand-receptor binding specificity is understood tomean a specific, saturable noncovalent interaction between the ligandand the receptor, and which is subject to competitive inhibition by asuitable competitor molecule. Preferred binding affinities (defined asthe amount of ligand required to fill one-half (50%) of availablereceptor binding sites) are described herein by dissociation constant(Kd). In one embodiment, preferred binding affinities of theligand-receptor complexes described herein have a Kd of less than 10⁻⁷M, preferably less than 5×10⁻⁷ M, more preferably less than 10⁻⁸ M. Inanother preferred embodiment, the receptor molecules have little or nosubstantial binding affinity for TGF-β.

As used herein, an "OP1-specific receptor analog" is understood to meana sequence variant of the ALK-2, ALK-3 or ALK-6 sequences which sharesat least 40%, preferably at least 45%, and most preferably at least 50%,amino acid identity in the extracellular ligand binding domain with thesequence defined by residues 23-122 of Seq. ID No. 7 (ALK-6), and whichhas substantially the same binding affinity for OP1 as ALK-2, ALK-3 orALK-6. ALK-6 and ALK-3 share 46% amino acid sequence identity in theirligand binding domains. Accordingly, in one preferred embodiment, theOP1-specific receptor analogs share at least 46% amino acid sequenceidentity with the extracellular, ligand binding domains of ALK-6 orALK-3.

As will be appreciated by those having ordinary skill in the art,OP1-specific receptor analogs also can have binding affinity for other,related morphogenic proteins. As used herein, an OP1-specific receptoranalog is understood to have substantially the same binding affinity forOP-1 as ALK-2, ALK-3 or ALK-6 if it can be competed successfully forOP-1 binding in a standard competition assay with a known OP-1 bindingreceptor, e.g., with ALK-2, ALK-3 or ALK-6. In one preferred embodiment,OP1-specific receptor analogs have a binding affinity for OP-1 definedby a dissociation constant of less than about 10⁻⁷ M, preferably lessthan about 5×10⁻⁷ M or 10⁻⁸ M. It is anticipated however, that analogshaving lower binding affinities, e.g., on the order of 10⁻⁶ M also willbe useful. For example, such analogs may be provided to an animal tomodulate availability of serum-soluble OP1 for receptor binding in vivo.Similarly, where tight binding interaction is desired, for example aspart of a cancer therapy wherein the analog acts as a ligand-receptorantagonist, preferred binding affinities may be on the order of 5×10⁻⁸M.

In another embodiment, the OP-1 binding receptor analogs contemplated bythe invention include proteins encoded by nucleic acids which hybridizewith the DNA sequence encoding the extracellular, ligand binding domainof ALK-2, ALK-3 or ALK-6 under stringent hybridization conditions, andwhich have substantially the same OP-1 binding affinity as ALK-2, ALK-3or ALK-6. As used herein, stringent hybridization conditions are asdefined in the art, (see, for example, Molecular Cloning: A LaboratoryManual, Maniatis et al., eds. 2d.ed., Cold Spring Harbor Press, ColdSpring Harbor, 1989.) An exemplary set of conditions is defined as:hybridization in 40% formamide, 5× SSPE, 5× Denhardt's Solution, and0.1% SDS at 37° C. overnight, and washing in 0.1× SSPE, 0.1% SDS at 50°C.

In still another embodiment, the OP-1 binding receptor analogscontemplated by the invention include part or all of a serine/threoninekinase receptor encoded by a nucleic acid that can be amplified with oneor more primers derived from ALK-1 (Seq. ID No. 1), ALK-2, ALK-3 orALK-6 sequence in a standard PCR (polymerase chain reaction)amplification scheme. In particular, a primer or, most preferably, apair of primers represented by any of the sequences of SEQ ID Nos. 12-15are envisioned to be particularly useful. Use of primer pairs (e.g.,SEQ. ID No. 12/15; 13/15; 14/15) are described in WO94/11502(PCT/GB93/02367).

Useful OP1-specific receptor analogs include xenogenic (foreign species)homologs of the murine and human ALK sequences described herein,including those obtained from other mammalian species, as well as other,eukaryotic, non-mammalian xenogenic homologs. Also contemplated arebiosynthetic constructs and naturally-occurring sequence variants ofALK-2, ALK-3 and ALK-6, provided these molecules, in all cases, sharethe appropriate identity in the ligand binding domain, and bind OP-1specifically as defined herein. In one embodiment, sequence variantsinclude receptor analogs which have substantially the same bindingaffinity for OP1 as ALK-2, ALK-3 or ALK-6 and which are recognized by anantibody having binding specificity for ALK-2, ALK-3 or ALK-6.

In another embodiment the receptors and OP-1 binding receptor analogscontemplated herein provide the means by which a morphogen, e.g., OP-1,can mediate a cellular response. In one embodiment these receptorsinclude ALK-2, ALK-3, or ALK-6, or sequence variants or OP-1 bindinganalogs thereof. In another embodiment, ALK-1, including sequencevariants thereof is contemplated to participate in an OP-1 mediatedcellular response.

OP1-specific receptor analogs may be used as OP1 antagonists. Forexample, a soluble form of a receptor, e.g., consisting essentially ofonly the extracellular ligand-binding domain, may be providedsystemically to a mammal to bind to soluble ligand, effectivelycompeting with ligand binding to a cell surface receptor, therebymodulating (reducing) the availability of free ligand in vivo for cellsurface binding.

The true tissue morphogenic proteins contemplated as useful receptorligands in the invention include OP-1 and OP-1 receptor-binding analogs.As used herein, an "OP-1 analog" or "OP-1 receptor-binding analog" isunderstood to include all molecules able to functionally substitute forOP-1 in Type I receptor binding, e.g., are able to successfully competewith OP-1 for receptor binding in a standard competition assay. In oneembodiment, useful OP-1 receptor-binding analogs include molecules whosebinding affinity is defined by a dissociation constant of less thanabout 5×10⁻⁶ M, preferably less than about 10⁻⁷ M or 5×10⁻⁷ M. As forthe OP-specific receptor analogs above, both stronger and weaker bindingaffinities are contemplated to be useful in particular applications. Inone preferred embodiment, these receptor-binding OP-1 analogs also bindOP-1 specific Type II serine/kinase receptors.

The OP-1 analogs contemplated herein, all of which mimic the bindingactivity of OP-1 or an OP-1-related protein sufficiently to act as asubstitute for OP-1 in receptor binding, can act as OP-1 agonists,capable of mimicking OP-1 both in receptor binding and in inducing atransmembrane effect e.g., inducing threonine or serine-specificphosphorylation following binding. Alternatively, the OP-1 analog canact as an OP-1 antagonist, capable of mimicking OP-1 in receptor bindingonly, but unable to induce a transmembrane effect, thereby blocking thenatural ligand from interacting with its receptor, for example. Usefulapplications for antagonists include their use as therapeutics tomodulate uncontrolled differentiated tissue growth, such as occurs inmalignant transformations such as in osteosarcomas or Paget's disease.

OP-1 analogs contemplated by the invention can be amino acid-based,e.g., sequence variants of OP-1, or antibody-derived sequences capableof functionally mimicking OP-1 binding to an OP-1-specific receptor.Examples of such antibodies may include anti-idiotypic antibodies. In aspecific embodiment, the anti-idiotypic antibody mimics OP1 both inreceptor binding and in ability to induce a transmembrane effect.Alternatively, the OP-1 analogs can be composed in part or in whole ofother chemical structures, e.g., the analogs can be comprised in part orin whole of nonproteinaceous molecules. In addition, the OP-1 analogscontemplated can be naturally sourced or synthetically produced.

As used herein, OP-1 related sequences include sequences sharing atleast 60%, preferably greater than 65% or even 70% identity with theC-terminal 102 amino acid sequence of OP-1 as defined in Seq ID NO.7,and which are able to substitute for OP-1 in ligand binding to ALK-2,ALK-3 or ALK-6, (e.g., able to compete successfully with OP-1 forbinding to one or more of these receptors.) OP-1 related sequencescontemplated by the invention include xenogenic homologs (e.g., theDrosophila homolog 60A), and the related sequences referenced herein andin the literature as OP-2, OP-3, BMP5, BMP6 (and its xenogenic homologVgr-1.) OP-1 related sequences also include sequence variants encoded bya nucleic acid which hybridizes with a DNA sequence comprising theC-terminal 102 amino acids of Seq. ID No. 9 under stringenthybridization conditions and which can substitute for OP1 in anOP1-receptor binding assay. In another embodiment, an OP1 sequencevariant includes a protein which can substitute for OP1 in aligand-receptor binding assay and which is recognized by an antibodyhaving binding specificity for OP1.

As used herein, "amino acid sequence homology" is understood to meanamino acid sequence similarity, and homologous sequences sharingidentical or similar amino acids, where similar amino acids areconserved amino acids as defined by Dayoff et al., Atlas of ProteinSequence and Structure; vol.5, Suppl.3, pp.345-362 (M. O. Dayoff, ed.,Nat'l BioMed. Research Fdn., Washington D.C. 1978.) Thus, a candidatesequence sharing 60% amino acid homology with a reference sequencerequires that, following alignment of the candidate sequence with thereference sequence, 60% of the amino acids in the candidate sequence areidentical to the corresponding amino acid in the reference sequence, orconstitute a conserved amino acid change thereto. "Amino acid sequenceidentity" is understood to require identical amino acids between twoaligned sequences. Thus, a candidate sequence sharing 60% amino acididentity with a reference sequence requires that, following alignment ofthe candidate sequence with the reference sequence, 60% of the aminoacids in the candidate sequence are identical to the corresponding aminoacid in the reference sequence.

As used herein, all receptor homologies and identities calculated useALK-6 as the reference sequence, with the extracellular domain referencesequence constituting residues 23-122 of Seq. ID No.7; and theintracellular serine/threonine kinase domain reference sequenceconstituting residues 206-495 of Seq. ID No.7. Similarly, all OP-1related protein homologies and identities use OP-1 as the referencesequence, with the C-terminal 102 amino acids described in Seq. ID No.constituting the seven cysteine domain.

Also as used herein, sequences are aligned for homology and identitycalculations as follows: Sequences are aligned by eye to maximizesequence identity. Where receptor amino acid extracellular domainsequences are compared, the alignment first maximizes alignment of thecysteines present in the two sequences, then modifies the alignment asnecessary to maximize amino acid identity and similarity between the twosequences. Where amino acid intracellular domain sequences are compared,sequences are aligned to maximize alignment of conserved amino acids inthe kinase domain, where conserved amino acids are those identified byboxes in FIG. 3. The alignment then is modified as necessary to maximizeamino acid identity and similarity. In all cases, internal gaps andamino acid insertions in the candidate sequence as aligned are ignoredwhen making the homology/identity calculation. Exemplary alignments areillustrated in FIGS. 2 and 3 where the amino acid sequences for theextracellular and intracellular domains, respectively are presented insingle letter format. In the figures "gaps" created by sequencealignment are indicated by dashes.

In one aspect, the invention contemplates isolated ligand-receptorcomplexes comprising OP-1 or an OP-1 analog as the ligand in specificbinding interaction with an OP-1 binding Type I receptor or receptoranalog, as defined herein. In another aspect, the invention contemplatesthe ligand-receptor complex comprises part or all of an OP-1 bindingType II receptor. Type II receptors contemplated to be useful includeType II receptors defined in the literature (referenced hereinabove) ashaving binding specificity for activin or a bone morphogenic proteinsuch as BMP-4. Such Type II receptors include daf4, ActRII and AtrII. Instill another aspect, the ligand-receptor complex comprises both a TypeI and a Type II receptor and OP1, or an OP1 analog as the ligand. In allcomplexes, the bound receptor can comprise just the extracellular,ligand binding domain, or can also include part or all of thetransmembrane sequence, and/or the intracellular kinase domain.Similarly, the OP-1 ligand may comprise just the receptor bindingsequence, longer sequences, including the mature dimeric species or anysoluble form of the protein or protein analog.

The OP-1 and OP-1 analogs described herein can interact specificallywith Type I and Type II receptors also known to interact with othermorphogenic proteins (e.g., BMP2/BMP4) and activin. Thus invention alsocontemplates the use of OP-1 and OP-1 receptor-binding analogs ascompetitors of specific BMP-receptor and activin-receptor interactions.As will be appreciated by those having ordinary skill in the art, thesebinding competitors may act as either agonists or antagonists (e.g., toinhibit an activin or BMP-mediated cellular response).

In another aspect, the invention contemplates binding partners havingspecific binding affinity for an epitope on the ligand-receptor complex.In a preferred embodiment, the binding partner can discriminate betweenthe complex and the uncomplexed ligand or receptor. In anotherembodiment, the binding partner has little or no substantial bindingaffinity for the uncomplexed ligand or receptor. In another preferredembodiment, the binding partner is a binding protein, more preferably anantibody. These antibodies may be monoclonal or polyclonal, may beintact molecules or fragments thereof (e.g., Fab, Fab', (Fab)'₂), or maybe biosynthetic derivatives, including, but not limited to, for example,monoclonal fragments, such as single chain F^(v) fragments, referred toin the literature as sF_(v) s, BABs and SCAs, and chimeric monoclonals,in which portions of the monoclonals are humanized (excluding thoseportions involved in antigen recognition (e.g., complementaritydetermining regions, "CDRs".) See, for example, U.S. Pat. Nos. 5,091,513and 5,132,405, the disclosures of which are incorporated herein byreference. Biosynthetic chimeras, fragments and other antibodyderivatives may be synthesized using standard recombinant DNAmethodology and/or automated chemical nucleic acid synthesis methodologywell described in the art and as described below.

In still another aspect, the invention provides molecules useful in thedesign and/or identification of receptor-binding morphogenic proteinanalogs as described below, as well as kits and methods, e,g., screeningassays, for identifying these analogs. The molecules useful in theseassays can include part or all of the receptor sequence of SEQ ID NO. 3,5 or 7, including amino acid sequence variants and OP-1 binding analogsand amino acid sequence variants thereof.

As described above, sequence variants are contemplated to havesubstantially the same binding affinity for OP-1 as the receptorsrepresented by the sequences in SEQ. ID Nos. 3-7. OP-1 binding receptoranalogs include other, known or novel Type I or Type II serine/threoninekinase receptors having binding affinity and specificity for OP-1 asdefined herein and which (1) share at least 40% amino acid identity withresidues 23-122 of Seq. ID No. 7, (2) are encoded by a nucleic acid thathybridizes under stringent conditions with a nucleic acid comprising thesequence defined by nucleotides 256-552 of Seq ID No. 7; or (3) areencoded by a nucleic acid obtainable by amplification with one or moreprimer sequences defined by Seq. ID Nos. 12-15. Currently preferred forthe assays of the invention are receptor sequences comprising at leastthe sequence which defines the extracellular, ligand binding domains ofthese proteins. The kits and assays may include just Type I receptors orboth Type I and Type II receptors. Similarly, the kits and screeningassays can be used in the design and/or identification of OP1-specificreceptor analogs. The OP-1 receptor-binding analogs and OP-1-bindingreceptor analogs thus identified then can be produced in reasonablequantities using standard recombinant expression or chemical synthesistechnology well known and characterized in the art. Alternatively,promising candidates can be modified using standard biological orchemical methodologies to, for example, enhance the binding affinity ofthe candidate analog as described in Example 10, below, and thepreferred candidate derivative then produced in quantity.

In still another aspect, the receptor and/or OP1-specific receptoranalogs can be used in standard methodologies for affinity purifyingand/or quantifying OP1 and OP1 analogs. For example, the receptor'sligand binding domain first may be immobilized on a surface of a well ora chromatographic column; ligand in a sample fluid then may be providedto the receptor under conditions to allow specific binding; non-specificbinding molecules then removed, e.g., by washing, and the bound ligandthen selectively isolated and/or quantitated. Similarly, OP1 and OP1analogs can be used for affinity purifying and/or quantifyingOP1-specific receptors and receptor analogs. In one embodiment, themethod is useful in kits and assays for diagnostic purposes which detectthe presence and/or concentration of OP1 protein or related morphogen ina body fluid sample including, without limitation, serum, peritonealfluid, spinal fluid, and breast exudate. The kits and assays also can beused for detecting and/or quantitating OP-1-specific receptors in asample.

In still another aspect the invention comprises OP1-specific receptorsand OP-1-binding receptor analogs useful in screening assays to identifyorgans, tissues and cell lines which express OP1-specific receptors.These cells then can be used in screening assays to identify ligandsthat modulate endogenous morphogen receptor expression levels, includingthe density of receptors expressed on a cell surface. Useful assaymethodologies may he modeled on those described in PCT US92/07359, andas described below.

The invention thus relates to compositions and methods for the use ofmorphogen-specific receptor sequences in diagnostic, therapeutic andexperimental procedures. Active receptors useful in the compositions andmethods of this invention can include truncated or full length forms, aswell as forms having varying glycosylation patterns. Active receptorsuseful in the invention also include chimeric constructs as describedbelow. Active OP1-specific receptors/analogs can be expressed fromintact or truncated genomic or cDNA, or from synthetic DNAs inprocaryotic or eucaryotic host cells, and purified, cleaved, refoldedand oxidized as necessary to form active molecules. Useful host cellsinclude prokaryotes, including E. coli and B. subtilis, and eukaryoticcells, including mammalian cells, such as fibroblast 3T3 cells, CHO,COS, melanoma or BSC cells, Hela and other human cells, theinsect/baculovirus system, as well as yeast and other microbial hostcell systems.

Thus, in view of this disclosure, skilled genetic engineers now can, forexample, identify and produce OP1-specific cell surface receptors oranalogs thereof; create and perform assays for screening candidate OP1receptor-binding analogs and evaluate promising candidates and theirprogency in therapeutic regimes and preclinical studies; modulate theavailability of endogenous morphogen for cell surface interactions;modulate endogenous morphogen-specific cell surface receptor levels;elucidate the signal transduction pathway induced by morphogen-cellsurface receptor binding; and modulate tissue morphogenesis in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the encoded ALK-2, ALK-3, ALK-6amino acid sequences, showing the signal sequence 10, the transmembranedomain 12, the extracellular ligand binding domain 14, and theintracellular serine/threonine kinase domain 16;

FIG. 2 is a homology alignment of the extracellular domains of ALK-2(SEQ ID NO:3), ALK-3 (SEQ ID NO:5), and ALK-6 (SEQ ID NO:7), aligned tomaximize amino acid identity, wherein conserved amino acids areidentified by boxes and conserved cysteines are indicated by asterisks;and

FIG. 3 is a homology alignment of the intracellular domain of ALK-2 (SEQID NO:3), ALK-3 (SEQ ID NO:5) and ALK-6 (SEQ ID NO:7), aligned tomaximize amino acid identity, wherein conserved amino acids are boxedand the serine/threonine kinase domain is indicated by arrows.

DETAILED DESCRIPTION

Disclosed herein are Type I and Type II receptors having bindingspecificity for true tissue morphogenic proteins, particularly OP1 andOP1-related proteins. It further has been determined that OP1 binds to abroader range of receptors than other known tissue morphogens or TGF-βfamily members. The Type I receptors disclosed herein, can be usedtogether with OP1 and OP1 analogs for therapeutic, diagnostic andexperimental uses as described herein below. Moreover, soluble forms ofthe OP1-binding receptor proteins, e.g., forms consisting essentially ofthe extracellular domain or a fragment thereof sufficient to bind OP1with specificity, can be used as a soluble therapeutic morphogenantagonist, as described below.

Following this disclosure, related OP1-specific receptors are available,as are high and medium flux screening assays for identifying OP1 analogsand OP1-specific receptor analogs. These analogs can be naturallyoccurring molecules, or they can be designed and biosyntheticallycreated using a rational drug design and an establishedstructure/function analysis. The analogs can be amino acid-based or canbe composed in part or whole of non-proteinaceous synthetic organicmolecules. Useful analogs also can include antibodies, preferablymonoclonal antibodies (including fragments thereof, e.g., Fab, Fab', and(Fab)'₂), or synthetic derivatives thereof, such as monoclonal singlechain F_(v) fragments known in the art as sF_(v) s, BABs, and SCAs (seebelow), and bispecific antibodies or derivatives thereof. When theseantibodies mimic the binding activity of OP-1 to a cell surface receptorwithout inducing the biological response OP-1 does upon binding, theantibody can compete for OP-1 binding and act as an antagonist. Theseantibodies or derivatives thereof also can mimic OP-1 both in receptorbinding and signal transduction, in which case the antibody acts as anOP-1 agonist. The antibodies and derivatives also can be used forinducing the morphogenic cellular response by crosslinking receptors tomorphogenic proteins, particularly OP1 and OP1-related proteins to formeither homo- or hetero-complexes of the Type I and Type II receptors.

The OP1-binding receptor sequences described herein (ALK2, ALK3, ALK6)also can be used to create chimeric sequences, wherein, for example,part or all of either the extracellular domain or the intracellulardomain is a non-ALK sequence or is created from two or more ALKsequences. These chimeric receptors can be synthesized using standardrecombinant DNA methodology and/or automated chemical nucleic acidsynthesis methodology well described in the art and as disclosed below.Chimerics can be used, for example, in OP1 analog assays, wherein theOP1-binding extracellular domain is coupled to a non-ALK intracellulardomain that is well characterized and/or readily detectable as a secondmessenger response system, as described below. Chimerics also can beused, for example, in high flux OP1 analogs screens and as part ofpurification protocols, wherein a soluble ligand binding domain of anOP1-specific receptor is immobilized onto a support e.g., by covalent ornon-covalent interactions, with a chromatographic matrix or the wellsurface of a 96-well plate. When immobilized onto a chromatographicmatrix surface, the receptor fragment can be used in a protocol toisolate OP1 or OP1 analogs. When immobilized on a well surface thereceptor fragment is particularly useful in a screening assay toidentify receptor-binding OP1 analogs in a standard competition assay.

The true tissue morphogenic proteins contemplated to be useful in themethods and compositions of the invention include forms having varyingglycosylation patterns and varying N-termini. The proteins can benaturally occurring or biosynthetically derived, and can be produced byexpression of recombinant DNA in prokaryotic or eukaryotic host cells.The proteins are active as a single species (e.g., as homodimers), orcombined as a mixed species. Useful sequences and eucaryotic andprocaryotic expression systems are well described in the art. See, forexample, U.S. Pat. Nos. 5,061,911 and 5,266,683 for useful expressionsystems.

Particularly contemplated herein are OP1 and OP1-related sequences.Useful OP1 sequences are recited in U.S. Pat. Nos. 5,011,691; 5,018,753and 5,266,683; in Ozkaynak et al. (1990) EMBO J 9:2085-2093; and Sampathet al. (1993) PNAS 90: 6004-6008. OP-1 related sequences includexenogenic homologs, e.g.; 60A, from Drosophila, Wharton et al. (1991)PNAS 88:9214-9218; and proteins sharing greater than 60% identity withOP1 in the C-terminal seven cysteine domain, preferably at least 65%identity. Examples of OP-1 related sequences include BMP5, BMP6 (and itsspecies homolog Vgr-1, Lyons et al. (1989) PNAS 86:4554-4558), Celeste,et al. (1990) PNAS 87:9843-9847 and PCT international applicationWO93/00432; OP-2 (Ozkaynak et al. (1992) J.Biol. Chem. 267:13198-13205)and OP-3 (PCT international application WO94/06447). As will beappreciated by those having ordinary skill in the art, chimericconstructs readily can be created using standard molecular biology andmutagenesis techniques combining various portions of differentmorphogenic protein sequences to create a novel sequence, and theseforms of the protein also are contemplated herein.

A particularly preferred embodiment of the proteins contemplated by theinvention includes proteins whose amino acid sequence in thecysteine-rich C-terminal domain has greater than 60% identity, andpreferably greater than 65% identity with the amino acid sequence of OPS(OP-1 sequence defining the C-terminal conserved six ctsteines, e.g.,residues 335-431 of Seq. ID No. 9).

In another preferred aspect, the invention contemplates osteogenicproteins comprising species of polypeptide chains having the genericamino acid sequence herin referred to as "OPX" which accommodates thehomologies between athe various ddentified species of the osteogenic OP1and OP2 proteins, and which is described by the amino acid sequencepresented below and in sequence ID No. 11. ##STR1## and wherein and Xaaat res. 2=(Lys or Arg); Xaa at res. 3=(Lys or Arg); Xaa at res. 11=(Argor Gln); Xaa at res. 16=(Gln or Leu); Xaa at res. 19=(Ile or Val); Xaaat res. 23=(Glu or Gln); Xaa at res. 26=(Ala or Ser); Xaa at res.35=(Ala or Ser); Xaa at res. 39=(Asn or Asp); Xaa at res. 41=(Tyr orCys); Xaa at res. 50=(Val or Leu); Xaa at res. 52=(Ser or Thr); Xaa atres. 56=(Phe or Leu); Xaa aat res. 57=(Ile or Met); Xaa at res. 58=(Asnor Lys); Xaa at res. 60=(Glu, Asp or Asn); Xaa at res. 61=(Thr, Ala orVal); Xaa at res, 65=(Pro or Ala); Xaa at res. 71=(Gln or Lys); Xaa atres. 73=(Asn or Ser); Xaa at res. 75=(Ile or Thr); Xaa at res. 80=(Pheor Tyr); Xaa at res. 82=(Asp or Ser); Xaa at res. 84=(Ser or Asn); Xaaat res. 89=(Lys or Arg); Xaa at res. 91=(Tyr or His); and Xaa at res.97=(Arg or Lys).

In still another preferred aspect, the invention contemplates osteogenicproteins encoded by nucleic acids which hybridize to DNA or RNAsequences encoding the C-terminal seven cysteine domain of OP1 or OP2under stringent hybridization conditions.

A brief description of the various terms of OP-1 useful in the inventionis described below:

OP1--Refers generically to the family of osteogenically active proteinsproduced by expression of part or all of the hOP1 gene. Also referred toin related applications as "OPI" and "OP-1".

OP1-PP--Amino acid sequence of human OP1 protein (prepro form), Seq. IDNo. 9, residues 1-431. Also referred to in related applications as"OP1-PP" and "OPP".

OP1-18Ser--Amino acid sequence of mature human OP1 protein, Seq. ID No.9, residues 293-431. N-terminal amino acid is serine. Originallyidentified as migrating at 18 kDa on SDS-PAGE in COS cells. Depending onprotein glycosylation pattern in different host cells, also migrates at23 kDa, 19 kDa and 17 kDa on SDS-PAGE. Also referred to in relatedapplications as "OP1-18."

OP1-16Ser; OP1-16Ala; OP1-16 Met; OP1-16 leu; OP1-16Val--N-terminallytruncated mature human OP1 protein species defined, respectively, byresidues 300-431; 316-431; 315-431; 313-431 and 318-431.

OPS--Amino acid sequence defining the C-terminal six cysteine domain,residues 335-431 of Seq. ID No. 9.

OP7--Amino acid sequence defining the C-terminal seven cysteine domain,residues 330-431 of Seq. ID No. 9.

Soluble form OP1--mature dimeric OP1 species having one or, preferablytwo copies of pro domain, e.g., at least residues 158-292 of Seq. ID No.9, preferably residues 48-292 or 30-292, non-covalently complexed withthe dimer.

The cloning procedure for obtaining OP1-binding ALK nucleic acidsequences, means for expressing receptor sequences, as well as othermaterial aspects concerning the nature and utility of these sequences,including how to make and how to use the subject matter claimed, will befurther understood from the following, which constitutes the best modecurrently contemplated for practicing the invention.

EXAMPLE 1 Identification of ALK-1, ALK-2, ALK-3 and ALK-6

The cloning and characterization of ALK-1, -2, -3, and -6 receptors aredescribed in detail in ten Dijke et al. (1993) Oncogene 8:2879-2887; and(1994) Science 264:101-104. The general structures of these proteins isdescribed in FIG. 1, and the sequence alignments between the ALK genesare shown in FIGS. 2 and 3. These molecules have similar domainstructures: an N-terminal predicted hydrophobic signal sequence (vonHeijne (1986) Nucl. Acids Res. 14: 4683-4690) is followed by arelatively small extracellular cysteine-rich ligand binding domain, asingle hydrophobic transmembrane region (Kyte & Doolittle (1982) J. Mol.Biol. 157, 105-132) and a C-terminal intracellular portion, whichconsists almost entirely of a kinase domain (FIG. 3).

The extracellular domains of these receptors, defined essentially byresidues 22-118 (SEQ. ID No. 1 ) for ALK-1; residues 16-123 (SEQ ID No.3) for ALK-2; residues 24-152 (SEQ. ID No. 5) for ALK-3; and residues23-122 (SEQ ID No. 7) for ALK-6, have cysteine-rich regions, butsequence similarity varies among the proteins. For example, ALK-3 andALK-6 share a high degree of sequence similarity in their extracellulardomains (46% identity) whereas ALK-2 shows less similarity with ALK 3 orALK6 (see FIG. 2.)

The positions of many of the cysteine residues in these receptors can bealigned, indicating that the extracellular domains likely adopt asimilar structural configuration.

The intracellular domains of these receptors are characterized by aserine/threonine kinase, defined essentially by residues 204-494 (SEQ.ID. No. 1) for ALK-1; residues 210-510 (SEQ ID No. 3) for ALK-2;residues 236-527 (SEQ ID No. 5) for ALK-3; and residues 206-497 (SEQ IDNo. 7) for ALK-6. The catalytic domains of kinases can be divided into12 subdomains with stretches of conserved amino-acid residues. The keymotifs are found in serine/threonine kinase receptors indicating thatthey are functional kinases. The consensus sequence for the binding ofATP (Gly-X-Gly-X-X-Gly (SEQ ID NO:16) in subdomain I followed by a Lysresidue further downstream in subdomain II) is found in all the ALKS.Moreover, ALK-1, ALK-2, ALK-3 and ALK-6 have the sequence motifs orsimilar motifs HRDLKSKN (SEQ ID NO:17) (Subdomain VIB) and GTKRYMAPE(SEQ ID NO:18) (Subdomain VIII), that are found in most of theserine/threonine kinase receptors and can be used to distinguish themfrom tyrosine kinase receptors. Two short inserts in the kinase domain(between subdomain VIA and VIB and between X and XI are unique tomembers of this serine/threonine kinase receptor family. In theintracellular domain, these regions, together with the juxtamembranepart and C-terminal tail, are the most divergent between family members.

Type II serine/threonine kinase receptors known in the art are describedand referenced herein above.

EXAMPLE 2 Receptor Expression

A. General Considerations

Receptor DNA, or a synthetic form thereof, can be inserted, usingconventional techniques well described in the art (see, for example,Maniatis (1989) Molecular Cloning A Laboratory Manual), into any of avariety of expression vectors and transfected into an appropriate hostcell to produce recombinant protein polypeptide chains, including bothfull length and truncated forms thereof. Shortened sequences, forexample, can be used for the production of soluble receptor fragments.

Useful host cells include E. coli, Saccharomyces cerevisiae, Pichiapastoris, the insect/baculovirus cell system, myeloma cells, and variousother mammalian cells. The full length forms of the proteins of thisinvention preferably are expressed in mammalian cells, as disclosedherein. Soluble forms may be expressed from both mammalian or bacterialcell systems. The vector additionally may include various sequences topromote correct expression of the recombinant protein, includingtranscription promoter and termination sequences, enhancer sequences,preferred ribosome binding site sequences, preferred mRNA leadersequences, preferred protein processing sequences, preferred signalsequences for protein secretion, and the like. The DNA sequence encodingthe gene of interest also may be manipulated to remove potentiallyinhibiting sequences or to minimize unwanted secondary structureformation. The recombinant morphogen receptor also may be expressed as afusion protein. After translation, the protein may be purified from thecells themselves or recovered from the culture medium. The DNA also mayinclude sequences which aid in expression and/or purification of therecombinant protein. One useful sequence for example, is a hexa-His(His₆) sequence, which adds a histidine tail to allow affinitypurification of the protein on an IMAC Cu2+ column (see below.)

For example, the DNA encoding the extracellular domain may be insertedinto a suitable expression vector for transformation into a prokaryotehost such as E. coli or B. subtilis, to produce a soluble, morphogenbinding fragment. The DNA may expressed directly or may be expressed aspart of a fusion protein having a readily cleavable fusion junction. Anexemplary protocol for prokaryote expression using MR-1 DNA is providedbelow. Recombinant protein is expressed in inclusion bodies and may bepurified therefrom using the technology disclosed in U.S. Pat. No.5,013,653, for example.

The DNA also may be expressed in a suitable mammalian host. Useful hostsinclude fibroblast 3T3 cells, (e.g., NIH 3T3, from CRL 1658) COS (simiankidney ATCC, CRL-1650) or CHO (Chinese hamster ovary) cells (e.g.,CHO-DXB11, from Lawrence Chasin, Proc. Nat'l. Acad. Sci. (1980)77(7):4216-4222), minklung epithelial cells (MV1Lu), human foreskinfibroblast cells, human glioblastoma cells, and teratocarcinoma cells.Other useful eukaryotic cell systems include yeast cells, theinsect/baculovirus system or myeloma cells.

To express an OP1-specific cell surface receptor, the DNA is subclonedinto an insertion site of a suitable, commercially available vectoralong with suitable promoter/enhancer sequences and 3' terminationsequences. Useful promoter/enhancer sequence combinations include theCMV promoter (human cytomegalovirus (MIE) promoter) present, forexample, on pCDM8, as well as the mammary tumor virus promoter (MMTV)boosted by the Rous sarcoma virus LTR enhancer sequence (e.g., fromClontech, Inc., Palo Alto). A useful induceable promoter includes, forexample, A Zn²⁺ induceable promoter, such as the Zn²⁺ metallothioneinpromoter (Wrana et al. (1992) Cell 71:1003-1014.) Other induceablepromoters are well known in the art and can be used with similarsuccess. Expression also may be further enhanced using transactivatingenhancer sequences. The plasmid also preferably contains an amplifiablemarker, such as DHFR under suitable promoter control, e.g., SV40 earlypromoter (ATCC #37148). Transfection, cell culturing, gene amplificationand protein expression conditions are standard conditions, well known inthe art, such as are described, for example in Ausubel et al., ed.,Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).Briefly, transfected cells are cultured in medium containing 5-10%dialyzed fetal calf serum (FCS), and stably transfected high expressioncell lines obtained by amplification and subcloning and evaluated bystandard Western and Northern blot analysis. Southern blots also can beused to assess the state of integrated receptor sequences and the extentof their copy number amplification.

The expressed protein then is purified using standard procedures. Acurrently preferred methodology uses an affinity column, such as aligand affinity column or an antibody affinity column, the boundmaterial then washed, and receptor molecules selectively eluted in agradient of increasing ionic strength, changes in pH or addition of milddenaturants. Alternatively, where a useful anchor sequence has beenadded to the DNA, such as a (His)₆ sequence, the column may be astandard affinity column such as Cu²⁺ IMAC column. Here, for example,the cell culture media containing the recombinant protein is passed overa Cu²⁺ IMAC column (for example, prepared with 25 mM imidazole). Thebound protein then is washed with a compatible solution and eluted withEDTA. The anchor sequence can be removed by a standard chemical orenzymatic procedure.

Mammalian cell expression is preferred where morphogen receptorexpression on a cell surface is desired. For example, cell surfaceexpression may be desired to test morphogen or morphogen analog bindingspecificity for a cell surface receptor under in vivo conditions. Cellsurface expression also may be most efficacious for medium flux cellularscreen assays as described below.

B.1 Exemplary Mammalian Cell Culture

The receptors tested in Examples 8 and 9 described below were expressedin (1) COS-1 cells; (2) mink lung epithelial cells (Mv1Lu); (3) AG1518human foreskin fibroblasts; (4) MG-63 human osteosarcoma cells; (5) PC12rat pheochromocytoma cells (all obtained from American Type CultureCollection, Rockville, Md.); (6) U-1240 MG human glioblastoma cells(Bengt Westermark, et al. (1988) Cancer Research 48:3910-3918); (7)Tera-2 teratocarcinoma cells (clone 13, Thompson et al. (1984) J CellSci 72:37-64); (8) MC3T3-El cells (Sudo et al. (1983) J. Cell Biol.96:191-198, and (9) ROS 17/2.8 rat osteosarcoma cells (Majeska et al.(1985) Endocrinology 116:170-179. The ROS cells were cultured in Ham'sF12 medium containing 14 mM HEPES buffer, 2.5 mM L-glutamine, 1.1 mMCaCl₂, 5% fetal bovine serum and antibiotics; MC3T3-E1 cells werecultured in a-MEM with 10% fetal bovine serum and antibiotics, andTera-2 cells were cultured in 5% CO₂ atmosphere at 37° C. in a-MEMcontaining 10% fetal bovine serum, 100 units/ml of penicillin and 50mg/ml of streptomycin, using tissue culture dishes pretreated with 0.1%swine skin gelatin (Sigma) in phosphate-buffered saline. Unlessotherwise specified, cells were cultured in DMEM containing 10% fetalbovine serum and antibiotics.

B.2 Transfection of cDNA

The receptors tested in Example 8.1 (ALK 1-6, daf-4) were transfected asfollows. Transient expression plasmids of ALK-1 to -6 and daf-4 weregenerated by subcloning into an expression vector (pSV7d, Truett et al.(1985) DNA 4:333-349) or into the pcDNA I expression vector (Invitrogen,San Diego). For transient transfection, COS-1 cells were transfectedwith 10 mg each of plasmids by a calcium phosphate precipitation methodusing a mammalian transfection kit (Stratagene, La Jolla), following themanufacturer's protocol. One day after transfection, the cells were usedfor the affinity labeling and cross-linking experiments.

EXAMPLE 3 Antibody Production

A. General Considerations

Antibodies capable of specifically binding the receptor molecules,ligand molecules, or the ligand-receptor complex itself, useful asanalogs and useful in immunoassays and in the immunopurification ofmorphogen receptors described may be obtained as described below.

Where antibodies specific to the OP1-specific receptors are desired, butwhich do not interfere with ligand binding, the antigenic sequencepreferably comprises the juxtamembrane sequence. Where antibodiescapable of competing for ligand binding are desired, the ligand bindingdomain may be used as the antigen source. Where antibodies to thecomplex are desired, the complex itself preferably is used as theantigenic sequence and candidate antibodies then tested for crossreactivity with uncomplexed ligand and receptors versus theligand-receptor complex. Finally, bispecific antibodies may used tocomplex ligand to a cell surface receptor (Type I or Type II) and/or totarget an agent or ligand to cells or tissue expressing a Type I or TypeII morphogen-specific receptor. Preferred bispecific antibody derivedmolecules are single chain binding sites described in U.S. Pat. Nos.5,091,513 and 5,132,405, the disclosures of which are incorporatedhereinabove by reference.

Antibodies useful as OP1 receptor-binding analogs may be obtained usingthe receptor ligand binding domain as the immunogen source and testingreceptor-binding analogs for their ability to compete with OP1 in acompetition binding assay. Similarly, where antibodies useful asOP1-specific receptor analogs are desired, OP1 is the immunogen sourceand the antibody candidate tested in a competition assay with receptorprotein.

Polyclonal antibodies specific for a morphogen receptor of interest maybe prepared generally as described below. Each rabbit is given a primaryimmunization (e.g., 500 mg) of antigen in 0.1% SDS mixed with 500 mlComplete Freund's Adjuvant. The antigen is injected intradermally atmultiple sites on the back and flanks of the animal. The rabbit isboosted after a month with 500 mg of antigen in the same manner usingincomplete Freund's Adjuvant. Test bleeds are taken from the ear veinseven days later. Two additional boosts and test bleeds are performed atmonthly intervals until antibody against the antigenic sequence isdetected in the serum using a standard Western blot. Then, the rabbit isboosted monthly with 100 mg/ml of antigen and bled (15 ml per bleed) atdays seven and ten after boosting.

Similarly, monoclonal antibodies specific for a given morphogen receptormolecule of interest may be prepared as described below: A mouse isgiven two injections of the antigenic sequence. The protein preferablyis recombinantly produced. Where it is desired that the antibodyrecognize an epitope on the morphogen binding surface of a receptor anantigenic fragment derived from the extracellular domain preferably isprovided. The first injection contains 100 mg of antigen in completeFreund's adjuvant and is given subcutaneously. The second injectioncontains 50 mg of antigen in incomplete adjuvant and is givenintraperitoneally. The mouse then receives a total of 230 mg of antigenin four intraperitoneal injections at various times over an eight monthperiod. One week prior to fusion, the mouse is boosted intraperitoneallywith antigen (e.g., 100 mg) and may be additionally boosted with anantigen-specific peptide conjugated to bovine serum albumin with asuitable crosslinking agent. This boost can be repeated five days (IP),four days (IP), three days (IP) and one day (IV) prior to fusion. Themouse spleen cells then are fused to commercially available myelomacells at a ratio of 1:1 using PEG 1500 (Boehringer Mannheim, Germany),and the fused cells plated and screened for ALK-specific antibodies,e.g., using ALK-2, ALK-3 or ALK-6 as antigen. The cell fusion andmonoclonal screening steps readily are performed according to standardprocedures well described in standard texts widely available in the art.(See, for example, Guide to Protein Purification Murray P. Deutscher,ed., Academic Press, San Diego, 1990.

B. Exemplary ALK-Specific Antisera

Antibodies used in the assay of Example 8 were obtained as follows.Rabbit antisera against ALK-1 to -6 were made against synthetic peptidescorresponding to the divergent, intracellular juxtamembrane parts.(ALK-1: residues 119-141; ALK-2: residues 151-172; ALK-3 residues181-202; ALK-6: residues 151-168.) Peptides were synthesized with anApplied Biosystems 430 A Peptide Synthesizer using t-butoxycarbonylchemistry, and purified by reverse phase HPLC. The synthetic peptideswere coupled to keyhole limpet hemocyanin (Calbiochem-Behring) usingglutaraldehyde, as described by Gullick et al. (1985) EMBO J 4:2869-2877. The coupled peptides then were mixed with Freund's adjuvantand used to immunize rabbits using standard methodologies.

EXAMPLE 4 OP1-Receptor Binding Assays

Ligand binding specificity is determined by evaluating the ability of areceptor molecule to bind a specific ligand, and the ability of thatligand to compete against itself and other molecules which bind thereceptor. Useful ligands will have a binding affinity for a solublemorphogen receptor extracellular domain such that dissociation constant(Kd) is less than about 10⁻⁶ M, preferably less than 5×10⁻⁷ M. Wherestronger binding interaction is desired, preferred affinities aredefined by a Kd of 10⁻⁸ -10⁻⁹ M. OP1-related proteins are expected to beable to bind with specificity to multiple different receptor molecules,although likely with differing affinities.

Ligand binding specificity can be assayed as follows, essentiallyfollowing standard protocols well described in the art and disclosed,for example, in Legerski et al. (1992) Biochem. Biophys. Res. Comm183:672-679 and Frakar et al., (1978) Biochem. Biophys. Res.Comm.80:849-857. In the ligand binding assays, a ligand having a known,quantifiable affinity for a morphogen receptor molecule of interest islabelled, typically by radioiodination (¹²⁵ I), e.g., by chromogenic orfluorogenic labeling, or by metabolic labelling, e.g., ³⁵ S, andaliquots of cells expressing the receptor on their surface are incubatedwith the labelled ligand, in the presence of various concentrations ofunlabelled potential competitor ligand. In the assays described inExamples 8 and 9, below, this competitor typically is the candidatemorphogen analog or an aliquot from a broth or extract anticipated tocontain a candidate morphogen analog.

Alternatively, a crosslinking agent may be used to covalently link theligand to the bound receptor, and the crosslinked complex thenimmunoprecipitated with an antibody specific to the ligand, receptor, orcomplex. (See, Example 8.)

A standard, exemplary protocol for determining binding affinity isprovided below. Briefly, cells expressing a receptor on their cellsurface are plated into 35 mM dishes and incubated for 48 hours in DMEM(Dulbecco's modified Eagle medium) plus 10% fetal calf serum. Purifiedmorphogen, here, e.g., OP-1, or an OP1-analog is iodinated with Na¹²⁵ Iby chloramine T oxidation, preferably having a specific activity ofabout 50-100 mCi/mg, essentially following the protocol of Frolik et al.(1984) J. Biol. Chem. 595:10995-11000. Labelled morphogen then ispurified using standard procedures, e.g., chromatographically. Platedcells then are washed twice with physiologically buffered saline in thepresence of 0.1% BSA, and incubated at 22° C. in the presence of BSA,buffer and labelled morphogen (1 ng) and various concentrations (e.g.,0-10 mg/ml) of unlabelled competitor, e.g., unlabelled morphogen orcandidate ligand analogs. Following binding, cells are washed threetimes with cold buffer, solubilized in 0.5 ml of 0.5N NaOH, removed fromthe dish, and radioactivity determined by gamma or scintillationcounter. Data then are expressed as percent inhibition, where 100%inhibition of specific binding is the difference between binding in theabsence of competitor and binding in the presence of a 100-fold molarexcess of unlabelled morphogen. Binding parameters preferably aredetermined using a computer program such as LIGAND (Munsun et al. (1980)Anal. Biochem. 107:220-259.)

Where the receptor cell surface binding domain is to be provided as asoluble protein, the assay can be performed in solution, most readily asan immunoprecipitation assay. In currently preferred assays themorphogen molecule is labelled and incubated with unlabelled receptorand candidate morphogen analogs. Receptor-specific antibody then isprovided to the solution to precipitate the receptor-morphogen complexand the amount of labelled morphogen in the precipitated complexdetermined using standard detection means.

Where the receptor or ligand is to be used in an affinity isolationprotocol, the molecule preferably is immobilized on a surface,preferably a matrix surface over which sample fluid will flow, allowingthe ligand of interest to bind, at letting nonbinding components passthrough as effluent. The complex then can be removed intact or theligand selectively removed with a desired eluant.

4.1 Screening Assay Considerations

In the analog screening assays described in Example 9 below, thepreferred protocol for assaying ligand-receptor binding is a standardcompetition or radioimmunoassay (RIA). Here the OP1 is labelled and therelative binding affinity of a candidate OP1 analog ligand in a sampleis measured by quantitating the ability of the candidate (unlabelledligand analog) to inhibit binding of the labelled ligand (competitormorphogen) by the receptor. In performing the assay, fixedconcentrations of receptor and labelled morphogen are incubated in theabsence and presence of unknown samples containing candidate ligands.Sensitivity can be increased by preincubating the receptor withcandidate ligand before adding the labelled morphogen. After thelabelled competitor has been added, sufficient time is allowed foradequate competitor binding, and then free and bound labelled morphogenare separated, and one or the other is measured. Useful morphogen labelsinclude radioactive labels, chromogenic or fluorogenic labels, andconjugated enzymes having high turnover numbers, such as horseradishperoxidase, alkaline phosphatase, or b-galactosidase, used incombination with chemiluminescent or fluorogenic substrates. In the samemanner, OP1-specific receptor analogs can be assayed for their affinityfor OP1 in competition assays with labelled OP1 specific receptors.

Assays for evaluating a candidate OP1 receptor-binding analog's abilityto mimic OP-1 in signal-transduction across a membrane are exemplifiedin detail in Example 9.2, below. Briefly, the assay involves use of acell (1) known to express an OP-1-specific receptor; or (2) which can bemodified to express an OP-1-specific receptor, and/or (3) which caninduce an OP-1-mediated cellular response. In the assay, the ability ofa candidate analog to induce an OP-1-specific cellular response ismonitored. Numerous OP-1 responsive cells and OP-1-mediated induciblecellular and biochemical markers are known and described in the art.Alternatively, and as exemplified below, an OP-1 inducible reporter genesystem can be constructed and used to advantage in the assay.

4.2 Diagnostic Assay Considerations

The ability to detect morphogens in solution provides a valuable toolfor diagnostic assays, allowing one to monitor the level of morphogenfree in the body, e.g., in serum and other body fluids. For example,OP-1 has been detected in a number of different body fluids, includingserum and spinal fluid, including cerebro-spinal fluid, saliva, milk andother breast exudates. (See, for example, PCT US92/07432, PCTUS93/07231, WO94/06449).

As one example, OP-1 is an intimate participant in normal bone growthand resorption. Thus, soluble OP-1 is expected to be detected at higherconcentrations in individuals experiencing high bone formation, such aschildren, and at substantially lower levels in individuals withabnormally low rates of bone formation, such as patients withosteoporosis, aplastic bone disease, or osteopenia. Monitoring the levelof OP-1 in serum thus provides a means for evaluating the status of bonetissue and bone homeostasis in an individual, as well as a means formonitoring the efficacy of a treatment to regenerate damaged or lostbone tissue. Alternatively, the level of OP-1 in bone tissue can beassessed in a bone tissue biopsy.

Similarly, OP-1 and other morphogens have been identified in braintissue. In particular, OP-1 is expressed and/or localized in developingand adult rat brain and spinal cord tissue, in the hippocampus,substantia nigra and the adendema glial cells, as well as associatedwith astrocytes and the extracellular matrix surrounding neuronal cellbodies. (See, PCTUS93/07331). Thus, monitoring the level of OP-1 inspinal fluid or associated with a nerve tissue biopsy can provide meansfor evaluating the status of nerve tissue in an individual, as well asmeans for monitoring the efficacy of a nerve regeration or repairtherapy.

For serum assays, the serum preferably first is partially purified toremove some of the excess, contaminating serum proteins, such as serumalbumin. Preferably the serum is extracted by precipitation in ammoniumsulfate (e.g., 45%) such that the complex is precipitated. Furtherpurification can be achieved using purification strategies that takeadvantage of the differential solubility of soluble morphogen complex ormature morphogens relative to that of the other proteins present inserum. Further purification also can be achieved by chromatographictechniques well known in the art. The sample fluid then can be assayedfor OP1 using the OP1-specific receptor(s) and binding assays asdescribed herein.

For a tissue biopsy, cells can be collected and stained with a labelledOP-1-specific antibody or receptor molecule. Alternatively, the OP-1protein selectively can be extracted and quantitated as described above.

Morphogens useful in the binding/screening assays contemplated hereininclude the soluble forms of the protein, e.g., the mature dimericspecies complexed with one or two copies of the pro domain, the maturedimeric species alone, and truncated forms comprising essentially justthe C-terminal active domain.

EXAMPLE 5 Transmembrane Signal Induction Assays/OP1 Mimetics

The kinase activity of the intracellular domains of the OP1-specificreceptors can be tested in an autophophorylation assay as described byMathews et al. (PCT/US92/03825, published Nov. 26, 1992). Briefly, theDNA fragment encoding at least the intracellular kinase domain of anOP1-specific receptor is subcloned into pGEX-2T (Smith et al.(1988) Gene67:31-40) to create a fusion protein between the putative kinase domainand glutathione S-transferase (GST). The plasmid is introduced into E.coli and the expressed fusion protein purified using glutathioneaffinity chromatography. About 100-200 ng of fusion protein or purifiedGST then are incubated with 25 mCi (gp³² P) ATP in 50 mM tris, 10 mMMgCl₂ buffer for 30 minutes at 37° C. Products then are analyzed by gelelectrophoresis and autoradiography. The fusion protein, but not GSTalone, becomes phophorylated, indicating that the kinase domain isfunctional. Phosphoamino acid analysis then can be performed todetermine the predominant amino acid being phosphorylated. Similarassays can be performed using similar fusion constructs expressed inmammalian cells.

Various signaling transduction assays are provided in Example 9, below.An assay also can be developed for testing kinase activity transductionupon ligand binding using a ligand-induced kinase activity assay knownin the art. Here, the ability of OP-1 analog to induce phosphorylationupon binding to the receptor is tested.

See, for example, various assays for measuring ligand-induced kinaseactivity described by Accili et al. (1991) J. Biol.Chem. 266:434-439 andNakamura et al. (1992) J.Biol. Chem. 267: 18924-18928,. For example,ligand-induced kinase activity (e.g., receptor autophosphorylation) canbe measured in vitro by incubating purified receptor in the presence andabsence of ligand (here, OP1 or OP1 analog, e.g., 10⁻⁷ M) underconditions sufficient to allow binding of the ligand to the receptor,followed by exposure to ³² P-ATP (e.g., 100 mCi in the presence of 10 mMTris-HCl (pH 7.6), 10 mM MgCl₂, 10 mM MnCl₂, 1 mM dithiothreitol, 0.15MNaCl₂, 0.1% Triton X-100 and 3% glycerol) and the amount ofphosphorylation measured, e.g., by SDS polyacrylamide gelelectrophoresis and autoradiography following immmunoprecipitation withantiphosphoserine, antiphosphothreonine or antiphosphotyrosine antibody(e.g., commercially available or made using standard antibodymethodologies.) While a low level of autophosphorylation may be detectedin the absence of ligand, incubation with ligand is anticipated tosignificantly increase (e.g., 5-20 fold increase) the amount ofphosphorylation detected.

In another assay for detecting ligand-induced receptorautophosphorylation, involving intact cells, receptor DNA is transfectedinto a suitable host cell, e.g., a fibroblast, which then is grown understandard conditions to create a confluent monolayer of cells expressingthe receptor on their cell surface. On the day of the experiment, cellsare incubated with or without ligand (e.g., OP1 or OP1 analog, e.g.,10⁻⁷ M) at 37° C., and then quickly washed with a "stopping solution"containing ATP (e.g., 0.1M NaF, 4 mM EDTA, 10 mM ATP, 10 mM sodiumorthovanadate, 4 mM sodium pyrophosphate). The cells then are frozen ina dry ice/ethanol bath, solubilized and the receptorsimmunoprecipitated, e.g., with an antireceptor antibody, as describedherein. The immune complexes then are segregated, washed, separated bygel electrophoresis using standard procedures and transferred to amembrane for Western blot analysis using standard procedures.Phosphorylation of the receptor then can be visualized byimmunodetection with a suitable antibody (e.g., antiphosphoserine,antiphosphothreonine or antiphosphotyrosine), as described above. Thebound antibody (e.g., bound antiphosphoserine, antiphosphothreonine orantiphosphotyrosine) then can be detected with ¹²⁵ I labelled protein A,followed by autoradiography. The amount of phosphorylated receptordetected is anticipated to be significantly greater (5-20 fold increase)in receptors incubated with ligand than receptors exposed to ATP in theabsence of ligand.

Ligand-induced receptor phosphorylation of exogenous substratessimilarly can be assayed essentially using the methodology describedherein. Here, a suitable substrate (e.g., a synthetic polypeptidecontaining serine, threonine or tyrosine amino acids) is provided to thereceptor following ligand exposure and prior to incubation with ATP. Thesubstrate subsequently can be segregated by immunoprecipitation with anantibody specific for the substrate, and phosphorylation detected asdescribed above. As for autophosphorylation, the amount ofphosphorylated substrate detected following ligand incubation isanticipated to be greater than that detected for substrates exposed toreceptors in the absence of ligand.

Alternatively, a reporter gene construct can be created to assaytransmembrane signal induction. Here, the expression control elementsfor an OP-1 inducible protein marker is fused to the open reading framesequence for any reporter gene, and induction of the reporter geneexpression then assayed. Useful reporter genes include the luciferasegene or GAL4 as well as other, easily characterizable markers.

EXAMPLE 6 Chimeric Receptor Molecules

Chimeric receptor molecules, e.g., comprising an ALK or ALK analogextracellular and transmembrane region and, for example, part or all ofan intracellular domain from another, different receptor or anintracellular domain from a different cell surface molecule, may beconstructed using standard recombinant DNA technology and/or anautomated DNA synthesizer to construct the desired sequence. As will beappreciated by persons skilled in the art, useful junctions includesequences within the transmembrane region and/or sequences at thejunction of either the intracellular or the extracellular domains. Alsoenvisioned are chimers where the extracellular domain or theintracellular domains themselves are chimeric sequences.

Chimeric sequences are envisioned to be particularly useful in screeningassays to determine candidate binding ligands (e.g., OP1 analogs, seebelow), where the non-receptor intracellular domain provides a suitablesecond messenger response system that is easy to detect. Potentiallyuseful other second messenger response systems include those which, whenactivated, induce phosphoinositide hydrolysis, adenylate cyclase,guanylate cyclase or ion channels.

Chimeric receptor molecules have particular utility in gene therapyprotocols. For example, a population of cells expressing a chimericmorphogen receptor molecule on their surface and competent forexpressing a desired phenotype can be implanted in a mammal at aparticular tissue locus. By careful choice of the ligand binding domainused on these receptors a physician can administer to the individual amorphogen agonist capable of: (1) binding to the chimeric receptor aloneand (2) stimulating the proliferation and/or differentiation of theimplanted cells without affecting endogenous cell populations.

EXAMPLE 7 Considerations for Identifying Other OP1 Specific Recetors inNucleic Acid Libraries

Identification of ALK Type I receptors that can bind OP-1 allows one toidentify other morphogen receptor sequences in different species as wellas in different tissues. The OP1-binding ALK sequences themselves can beused as a probe or the sequence may be modified to account for otherpotential codon usage (e.g., human codon bias.) Currently preferredprobe sequences are those which encode the receptor's extracellulardomain.

Probes based on the nucleic acid sequence of Seq. ID Nos.1, 3, 5 or 7can be synthesized on commercially available DNA synthesizers, e.g.Applied Biosystems model 381A, using standard techniques, e.g. Gait,Oligonucleotide synthesis: A Practical Approach, (IRL Press, WashingtonD.C., 1984). It is preferable that the probes are at least 8-50 baseslong, more preferably 18-30 bases long. Probes can be labeled in avariety of ways standard in the art, e.g. using radioactive, enzymaticor colormetric labels as described, for example, by Berent et al,(May/June 1985) Biotechniques: 208-220; and Jablonski et al, (1986)Nucleic Acids Research 14: 6115-6128.

Preferably, low stringency conditions are employed when screening alibrary for morphogen receptor sequences using a probe derived fromOP1-binding receptor. Preferred ALK-specific probes are thosecorresponding to bases encoding the extracellular domain ("ECD"), orencoding a unique (nonhomologous) sequence within the cytoplasmicdomain. Useful probes may be designed from bases encoding thejuxtamembrane region, for example. The probe may be further modified touse a preferred species codon bias. Alternatively, probes derived fromthe serine/threonine kinase domain can be used to identify new membersof the receptor kinase family which can be screened for OP1 bindingusing the methods described in Example 8.

For example, for a probe of about 20-40 bases a typicalprehybridization, hybridization, and wash protocol is as follows: (1)prehybridization: incubate nitrocellulose filters containing thedenatured target DNA for 3-4 hours at 55° C. in 5× Denhardt's solution,6× SSC (20× SSC consists of 175g NaCl, 88.2 g sodium citrate in 800 mlH₂ O adjusted to pH. 7.0 with 10N NaOH), 0.1% SDS, and 100 mg/mldenatured salmon sperm DNA, (2) hybridization: incubate filters inprehybridization solution plus probe at 42° C. for 14-48 hours, (3)wash; three 15 minutes washes in 6× SSC and 0.1% SDS at roomtemperature, followed by a final 1-1.5 minute wash in 6× SSC and 0.1%SDS at 55° C. Other equivalent procedures, e.g. employing organicsolvents such as formamide, are well known in the art.

Alternatively, morphogen receptor-specific DNA can be amplified using astandard PCR (polymerase chain reaction) methodology such as the onedisclosed herein, to amplify approximately 500 base pair fragments. Asfor the hybridization screening probes described above, the primersequences preferably are derived from conserved sequences in theserine/threonine kinase domain. The primers disclosed herein, in Seq. IDNos. 12-15 are envisioned to be particularly useful, particularly incombination.

Examples of useful PCR amplifications, including the use of the primersrecited herein, are disclosed in ten Dijke, et al. (1993) Oncogene8:2879-2887 and (1994) Science 264:101-104, and which also describe theisolation protocols for ALK-1, ALK-2, ALK-3 and ALK-6.

7.1 TISSUE DISTRIBUTION OF MORPHOGEN RECEPTORS

Determining the tissue distribution of OP1-specific receptors can beused to identify tissue and cell sources which express these receptors,to identify new, related OP1-specific receptor molecules, as well as toidentify target tissues for OP1-receptor interactions under naturallyoccurring conditions. The OP-1 specific receptor molecules (or theirmRNA transcripts) readily are identified in different tissues usingstandard methodologies and minor modifications thereof in tissues whereexpression may be low. For example, protein distribution can bedetermined using standard Western blot analysis or immunohistologicaldetection techniques, and antibodies specific to the morphogen receptormolecules of interest. Similarly, the distribution of morphogen receptortranscripts can be determined using standard Northern hybridizationprotocols and transcript-specific probes or by in situ hybridization.

Any probe capable of hybridizing specifically to a transcript, anddistinguishing the transcript of interest from other related transcriptscan be used. Because the morphogen receptors described herein likelyshare high sequence homology in their intracellular domains, the tissuedistribution of a specific morphogen receptor transcript may best bedetermined using a probe specific for the extracellular domain of themolecule. For example, a particularly useful ALK-specific probe sequenceis one derived from a unique portion of the 5' coding sequence, thesequence corresponding to the juxtamembrane region, or the 5' or 3'noncoding sequences. The chosen fragment then is labelled using standardmeans well known and described in the art and herein.

Using these receptor-specific probes, which can be syntheticallyengineered or obtained from cloned sequences, receptor transcripts canbe identified and localized in various tissues of various organisms,using standard methodologies well known to those having ordinary skillin the art. A detailed description of a suitable hybridization protocolis described in ozkaynak, et al., (1991) Biochem. Biophys. Res. Commn.179:116-123, and Ozkaynak, et al. (1992) J Biol. Chem. 267:25220-25227.Briefly, total RNA is prepared from various tissues (e.g., murine embryoand developing and adult liver, kidney, testis, heart, brain, thymus,stomach) by a standard methodologies such as by the method ofChomczynski et al. ((1987) Anal. Biochem 162:156-159) and describedbelow. Poly (A)+ RNA is prepared by using oligo (dT)-cellulosechromatography (e.g., Type 7, from Pharmacia LKB Biotechnology, Inc.).Poly (A)+ RNA (generally 15 mg) from each tissue is fractionated on a 1%agarose/formaldehyde gel and transferred onto a Nytran membrane(Schleicher & Schuell). Following the transfer, the membrane is baked at80° C. and the RNA is cross-linked under UV light (generally 30 secondsat 1 mW/cm²). Prior to hybridization, the appropriate probe is denaturedby heating. The hybridization is carried out in a lucite cylinderrotating in a roller bottle apparatus at approximately 1 rev/min forapproximately 15 hours at 37° C. using a hybridization mix of 40%formamide, 5× Denhardts, 5× SSPE, and 0.1% SDS. Following hybridization,the non-specific counts are washed off the filters in 0.1× SSPE, 0.1%SDS at 50° C.

EXAMPLE 8 Demonstration That ALK-2, ALK-3 and ALK-6 are OP1-BindingReceptors

The tissue morphogenic proteins OP1 and BMP4 were tested for specificbinding interaction with the ALK receptors in receptor-transfected cells(where the receptor is over-expressed), and in nontransfected cells. Itpreviously was known that ALK-5 interacted specifically with TGFS1 andALK-2 and ALK-4 interacted specifically with activin. In the experiment,complexes were crosslinked and immuno-precipitated with an ALK-specificantibody as described below. To date, no binding with ALK-1 under theconditions of this protocol have been detected.

Binding and affinity cross-linking using disuccinimidyl suberate (PierceChemical Co.) were performed using standard methods (e.g., Franzen etal. (1993) Cell 75:681-692 and Ichijo et al. (1990) Exp. Cell Res.187:10995-11000.) A typical protocol is described below. Modificationsfrom this protocol for individual experiments were standard changesanticipated to produce the same result as for the recited procedure.Briefly, cells in multi-well plates were washed with binding buffer(e.g., phosphate buffered saline containing 0.9 mM CaCl₂, 0.49 mM MgCl₂and 1 mg/ml bovine serum albumin (BSA)), incubated on ice in the samebuffer with labelled ligand, in the presence and absence of excessunlabelled ligand for sufficient time for the reaction to equilibrate(e.g., 3 hours.) Cells were washed and the crosslinking was done in thebinding buffer without BSA together with 0.28 mM disuccinimidyl suberatefor 15 min on ice. Cells were harvested by addition of 1 ml ofdetachment buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10% glycerol, 0.3mM PMSF.) Cells then were pelleted by centrifugation, then resuspendedin 50 ml of solubilization buffer (125 mM NaCl, 10 mM Tris-HCl, pH 7.4,1 mM EDTA, 1% Triton X-100, 0.3 mM PMSF, 1% Trasylol) and incubated for40 minutes on ice. Cells were centrifuged again and supernatantssubjected to analysis by standard SDS-gel electrophoresis using 4%-15%polyacrylamide gels, followed by autoradiography.

Cell lysates obtained following affinity cross-linking via the generalprotocol described above were immunoprecipitated using antisera againstALKs (e.g., raised against the ALK juxtamembrane region), or directlyanalyzed by SDS-gel electrophoresis using gradient gels consisting of5-12% or 5-10% polyacrylamide. The gels were fixed and dried, and thensubjected to autoradiography or analysis using phosphorImager (MolecularDynamics).

8.1 Binding of OP-1 and/or BMP-4 to ALKs in Transfected Cells.

COS-1 cells transfected with ALK cDNA were tested for the binding of ¹²⁵I-OP-1 and ¹²⁵ I-BMP-4, in the presence or absence of co-transfectedType II receptor DNA: daf-4 cDNA or ActRII (Estevez et al. (1993) Nature365:644-649 and Attisano et al. (1992) Cell 68:97-108, disclosing theDNA sequence for these Type II receptors and the disclosure of which isincorporated herein by reference.) Since the cross-linked complexes weresometimes difficult to visualize because of high background, sampleswere immunoprecipitated by antisera against each ALK. The results arepresented in Table I below. In the Table, "N/T" means "not tested".Binding was specific as determined by standard competition assays. Thevalues represented by "+/-", "+", "++", "+++", and "-" are allqualitative descriptors of the relative amount of radioactivity measuredwhen the crosslinked molecules were gel electrophoresed and subjected toautoradiography. More radioactivity measured indicates a strongerbinding interaction detected. In the Table the strength of bindinginteraction is as follows: +++>++>+>+/->-.

                  TABLE I    ______________________________________    .sup.125 I OP1         .sup.125 I BMP4    +daf4     -daf4   ActRII         +daf4                                          -daf4                                               +ActRII    ______________________________________    ALK1   -      -       -     ALK1   -    -    N/T    ALK2   ++     +/-     ++    ALK2   -    -    N/T    ALK3   ++     -       +/-   ALK3   +++  ++   N/T    ALK4   -      -       -     ALK4   -    -    N/T    ALK5   -      -       -     ALK5   -    -    N/T    ALK6   +++    ++      +     ALK6   +++  +++  N/T    ______________________________________

In the absence of daf-4, OP-1 bound to ALK-6, whereas BMP-4 bound toALK-3 and ALK-6. Weaker binding of OP-1 to ALK-2 was also observed.Other ALKs did not bind OP-1 or BMP-4 in the absence of Daf-4. When ALKcDNAs were co-transfected with the daf-4 cDNA, increased binding of OP-1to ALK-2 and ALK-6 was seen. In addition, ALK-3 also was found to bindOP-1 in the co-transfected cells. Similarly, increased binding of BMP-4to ALK-3 and ALK-6 could be observed. Co-transfection of two differenttypes of ALKs did not further increase the binding of OP-1 or BMP-4. Incells co-transfected with the DNA for ActRII and ALK-2, ALK-3 or ALK-6,OP1-receptor binding was enhanced.

The sizes of the cross-linked complexes were slightly higher for ALK-3than for ALK-2 and ALK-6, consistent with its slightly larger size.Complexes of about 95 kDa as well as multiple components of 140-250 kDawere also co-immunoprecipitated with certain of the ALKs.

In standard competition assays performed with the Type I receptors inthe presence and absence of the Type II receptors, the binding of OP-1and BMP-4 could be competed with excess amounts of unlabeled OP-1,verifying the binding specificity of these interactions where theyoccurred.

These results demonstrate that ALK-2, ALK-3 and ALK-6 can serve as TypeI receptors for OP-1. Notably, ligand binding apparently can be enhancedin the presence of Type II receptors. Moreover, OP-1 is able to interactwith both a "bone morphogen" Type II receptor (daf 4) and an "activin"Type II receptor (ActRII), whereas, for example, activin only interactswith the ActRII Type II receptor. The data indicate that OP1 has abroader spectrum of receptor (Type I and Type II) binding affinitiesthan do other tissue morphogenic proteins, or other members of the TGF-βfamily. It is anticipated that OP1 will have specific bindinginteractions with other "activin-binding" or "bone morphogen-binding"Type II receptors.

The ability of OP-1 to bind to Type I, Type II receptors having bindingspecificity for activin or BMP4 but not TGF-B, indicates that OP-1 andOP-1 analogs will be useful as competitors of activin or BMP4 binding tocell surface receptors. In particular, OP-1 and OP-1 analogs will beuseful for competing with activin-ALK-2 binding and/oractivin-ALK-2/ActRII (or other Type II) receptor binding; and forcompeting with BMP4 (or BMP2 )-ALK-6 binding, and/or BMP2/4-ALK-6/daf 4(or other Type II) binding. The OP-1 competitors may act as antagonists(e.g., binding competitors unable to induce the signal transductioncascade upon binding) or as agonists (e.g., able both to bind and inducethe signal transduction cascade).

8.2 Identification of OP1-Specific Receptors in Nontransfected Cells

ALK-5 has been shown to bind TGF-β1, and ALKs 2, 4 bind activin A withhigh affinities in nontransfected cells (ten Dijke, Oncogene, (1993₋₋ ;Science, (1994) referenced herein above.) In the present experiment, thebinding affinity of OP1 and/or BMP4 to receptors in nontransfected cellswas demonstrated as follows. The results corroborate the transfectedcell data, verifying that OP1 interacts specifically with ALK-2, ALK-3and ALK-6, but not ALK-4 or ALK-5.

MC3T3-E1 osteoblasts are well characterized cells known to respond toOP-1 and BMP-4 in the induction of alkaline phosphatase activity(Paralkar (1991) PNAS 8: 3397-3401.) These cells were affinity labeledusing ¹²⁵ I-OP-1 as described herein above, and cross-linked complexesof about 75 kDa were seen, which were immunoprecipitated only with theALK-2 antiserum. Tera-2 teratocarcinoma cells and MvlLu cells respondedto OP-1 as measured by production of plasminogen activator inhibitor-1(PAI-1). Similar to MC3T3-E1 cells, cross-linked complexes using ¹²⁵I-OP-1 in Tera-2 teratocarcinoma were immunoprecipitated only by theALK-2 antiserum.

On the other hand, cross-linked complexes using ¹²⁵ I-OP-1 to Mv1Lucells were immunoprecipitated by ALK-2 as well as ALK-3 and ALK-6antisera. Mv1Lu cells are known to express ALK-4 and ALK-5 (Ebner (1993)Science 260:1344-1348), but cross-linked complexes with ¹²⁵ I-OP-1 werenot precipitated by antisera against these receptors. Similarly,cross-linked complexes in U-1240 MG glioblastoma cells wereimmunoprecipitated by ALK-2 and ALK-6 antisera, and weakly by ALK-3antiserum. In contrast, cross-linking of ¹²⁵ I-OP-1 to AG1518 humanforeskin fibroblasts yielded weak immunoprecipitated components only byALK-3 antiserum. Type II receptor-like components of about 95 kDa aswell as high molecular weight complexes of 140-250 kDaco-immunoprecipitated with certain ALKs in the Tera-2 cells, MvlLu cellsand U-1240 MG cells.

Receptors for BMP-4 have also been investigated using nontransfectedcells. Cross-linked complexes using ¹²⁵ I-BMP-4 to MC3T3-E1 cells andAG1518 human foreskin fibroblasts were immunoprecipitated only by ALK-3.On the other hand, cross-linking of ¹²⁵ I-BMP-4 to Tera-2 cells did notyield any immunoprecipitated components by antisera against ALKs.

¹²⁵ I-OP-1 and/or ¹²⁵ I-BMP-4 also were demonstrated by affinitycross-linking to interact specifically with receptors in otherBMP-responsive cells, e.g., MG-63 osteosarcoma cells and PC12pheochromocytoma cells. A summary of the binding of ALKs to OP-1 orBMP-4 in different cell types is shown in Table II, below. In the Table,"N/T" means not tested, and receptors presented in brackets indicatecomparatively lower quantities of radioactive complexes detected.

                  TABLE II    ______________________________________    Cell lines    Binding of OP-1                               Binding of BMP-4    ______________________________________    Mouse osteoblasts                  ALK-2        ALK-3    (MC3T3 -E1    Mink lung epithelial                  ALK-2, -3, -6                               N/T    cells (Mv1Lu)    Human glioblastoma                  ALK2,  -3!, -6                               N/T    Human teratocarcinoma                  ALK2         --    (Tera-2)    Human foreskin                   ALK3!       ALK3    fibroblasts (AG1518)    Rat osteosarcoma                  ALK2,  -3!   N/T.    (ROS17/2.8)    ______________________________________

EXAMPLE 9 OP1, OP1-SPECIFIC RECEPTOR ANALOG SCREENING ASSAYS

The present invention is useful to determine whether a ligand, such as aknown or putative drug, is capable of binding to and/or activating anOP1-specific cell surface receptor as described herein. Ligands capableof specific binding interaction with a given OP1-specific receptor(e.g., ALK-2, ALK-3, ALK-6 ) are referred to herein as OPi analogs andcan be used for therapeutic and diagnostic applications. Some analogswill have the ability to stimulate morphogenetic activity in the cell,mimicking both the receptor binding and signal transducing activity ofOP1. These are referred to OP1 agonists or mimetics. Others will havestrong binding affinity but will not stimulate morphogenesis, these areOP1 antagonists. The analogs can be amino acid-based, or they can becomposed of non-proteinaceous chemical structures.

The methods and kits described below similarly can be used to identifyaP1-specific receptor analogs, capable of mimicking the binding affinityof ALK-2, ALK-3 or ALK-6 for OP1. The analogs can be provided to amammal to interact with serum-soluble OP1, effectively sequestering theprotein and modulating its availability for cell surface interaction.

Transfection of an isolated clone encoding a morphogen receptor into thecell systems described above provides an assay system for the ability ofligands to bind to and/or to activate the receptor encoded by theisolated DNA molecule. Transfection systems, such as those describedabove, are useful as living cell cultures for competitive binding assaysbetween known or candidate drugs and ligands which bind to the receptorand compete with the binding of known morphogens, which are labeled byradioactive, enzymatic, spectroscopic or other reagents. Membranepreparations containing the receptor and isolated from transfected cellsare also useful in these competitive binding assays. Alternatively, andcurrently preferred, purified receptor molecules or their ligand bindingextracellular domains can be plated onto a microtiter well surface, in amodification of a sandwich assay, e.g., as a competition assay, such asan RIA, described above. Finally, as described above, solution assays,and using only the receptor extracellular domain, also may be used toadvantage in these assays. Functional assays of second messenger systemsor their sequelae in transfection systems act as assays for bindingaffinity and efficacy in the activation of receptor function or efficacyin the antagonism of receptor function. Such a transfection systemconstitutes a "drug discovery system", useful for the identification ofnatural or synthetic compounds with potential for drug development thatcan be further modified or used directly as therapeutic compounds toactivate or inhibit the natural functions of the receptor encoded by theisolated DNA molecule.

Once such candidate drugs (e.g., OP-1 or receptor-binding analogsthereof) are identified, they can be produced in reasonable, usefulquantities using standard methodologies known in the art. Aminoacid-based molecules can be encoded by synthetic nucleic acid molecules,and expressed in a recombinant expression system as described hereinabove or in the art. Alternatively, such molecules can be chemicallysynthesized, e.g., by means of an automated peptide synthesizer, forexample. Non-amino acid-based molecules can be produced by standardorganic chemical synthesis procedures. Where the candidate molecule isof undetermined structure, or composition, its composition readily canbe determined by, for example, mass spectroscopy. Two approaches toidentifying analogs typically are practiced in the art: high fluxscreens and rational design of ligand mimetics. High flux screenstypically screen naturally sourced materials or chemical banks for theirability to bind a protein of interest, here, e.g., the receptor.Typically, compounds are obtained from a range of sources, e.g.,chemical banks, microbial broths, plant and animal extracts, and thelike. In a high flux screen typically, purified receptor, preferably thesoluble, ligand binding extracellular domain, is plated onto amicrotiter well surface and a standard volume of a sample solution to betested then is added. Also added is a standard volume having a knownquantity of a purified ligand known to bind the receptor withspecificity. Preferably the ligand is labelled with a substance that isreadily detectable by automated means (e.g., radiolabel, chromophoric,fluorometric, enzymatic or spectroscopic label.) The wells then arewashed and the amount of label remaining after washing or the amount oflabel remaining associated with the receptor then is detected. Positivescores are identified by the ability of the test substance to preventinteraction of the labelled ligand with the receptor. The screeningassays can be performed without undue experimentation, using standardmolecular and cell biology tools in common use in the art. For example,screening assays can be performed in standard 96-well plates. Fifteensuch plates reasonably can be set up at a time to perform multiplescreening assays in parallel. Thus, with only 10-11 reiterations of thescreening assay 15,625 (5⁶) compounds can be screened for their bindingaffinity. Even allowing for a maximum incubation time of 2 hours, all15,625 compounds reasonably can be assayed in a matter of days.

High flux screens exploit both the high degree of specificity of thelabelled ligand for its receptor, as well as high throughput capacity ofcomputer driven robotics and computer handling of data. Candidateanalogs identified in this manner, then can be analyzed structurally andthis information used to design and to synthesize analogs havingenhanced potency, increased duration of action, increased selectivityand reduced side effects. Candidates also can be used in a rationaldesign program as described below. Finally, candidate analogs also canbe tested to determine morphogenetic effect, if any, as described below.

The second approach to the identification of analogs uses a rationaldesign approach to create molecules capable of mimicking the bindingeffect of OP1 with an OP1-specific receptor. Here the relevant structurefor receptor binding is analyzed to identify critical sequences andstructures necessary for binding activity and this information can beused to design and synthesize minimal size morphogen analogs. As forcandidate compounds in the high flux assay, design candidates can betested for receptor binding activity as described above. As describedabove, a candidate sequence can be further modified by, for examplestandard biological or chemical mutagenesis techniques to create acandidate derivative having, for example, enhanced binding affinity oranother preferred characteristic.

Antibodies capable of interacting specifically with the receptor andcompeting with OP1 binding also can be used as an analog. Antibodies canbe generated as described above.

OP1 analogs may be evaluated for their ability to mimic OP1 or toinhibit OP1 binding (e.g., agonists or antagonists) by monitoring theeffect of the analogs on cells bearing an OP1-specific receptor (e.g.,ALK-2, ALK-3 or ALK-6.) OP1 agonists are anticipated to have utility inany application where tissue morphogenesis is desired, such as in theregeneration of damaged tissue resulting from mechanical or chemicaltrauma, degenerative diseases, tissue destruction resulting from chronicinflammation, cirrhosis, inflammatory diseases, cancer and the like, andin the regeneration of tissues, organs and limbs. OP1 antagonists areenvisioned to have utility in applications where tissue morphogenesis isto be limited as, for example, in the treatment of malignanttransformations including, but not limited to, osteosarcomas and Paget'sdisease.

Several exemplary systems for assaying the ability of a candidate analogtransduce an OP-1-specific signal across the cellular membrane aredescribed below.

9.1 Induction of Osteoblast Differentiation Markers

For example, OP1 is known to preferentially induce differentiation ofprogenitor cells, including embryonic mesenchymal cells and primaryosteoblasts (see, for example, PCT US92/07432) As one example, OP1analogs can be tested for their ability to induce differentiation ofprimary osteoblasts, by measuring the ability of these analogs to induceproduction of alkaline phosphatase, PTH-mediated cAMP and osteocalcin,all of which are induced when primary osteoblasts are exposed to OP-1,60A or DPP.

Briefly, the assays may be performed as follows. In this and allexamples involving osteoblast cultures, rat osteoblast-enriched primarycultures preferably are used. Although these cultures are heterogeneousin that the individual cells are at different stages of differentiation,these cultures are believed to more accurately reflect the metabolismand function of osteoblasts in vivo than osteoblast cultures obtainedfrom established cell lines. Unless otherwise indicated, all chemicalsreferenced are standard, commercially available reagents, readilyavailable from a number of sources, including Sigma Chemical, Co., St.Louis; Calbiochem, Corp., San Diego and Aldrich Chemical Co., Milwaukee.

Rat osteoblast-enriched primary cultures are prepared by sequentialcollagenase digestion of newborn suture-free rat calvaria (e.g., from1-2 day-old animals, Long-Evans strain, Charles River Laboratories,Wilmington, Mass.), following standard procedures, such as aredescribed, for example, in Wong et al., (1975) PNAS 72:3167-3171. Ratosteoblast single cell suspensions then are plated onto a multi-wellplate (e.g., a 24 well plate) at a concentration of 50,000 osteoblastsper well in alpha MEM (modified Eagle's medium, Gibco, Inc., LongIsland) containing 10% FBS (fetal bovine serum), L-glutamine andpenicillin/streptomycin. The cells are incubated for 24 hours at 37° C.,at which time the growth medium is replaced with alpha MEM containing 1%FBS and the cells incubated for an additional 24 hours so that the cellsare in serum-deprived growth medium at the time of the experiment.

Alkaline Phosphatase Induction of Osteoblasts

The cultured cells in serum-free medium are incubated with OP1, OP1analog or a negative control, using a range of concentrations. Forexample, 0.1, 1.0, 10.0, 40.0 or 80.0 ng OP-1/ml medium typically areused. 72 hours after the incubation period the cell layer is extractedwith 0.5 ml of 1% Triton X-100. The resultant cell extract then, iscentrifuged, and 100 ml of the extract is added to 90 ml ofparanitrosophenylphospate (PNPP)/glycerine mixture and incubated for 30minutes in a 37° C. water bath and the reaction stopped with 100 mlNaOH. The samples then are run through a plate reader (e.g., DynatechMR700 plate reader, and absorbance measured at 400 nm, usingp-nitrophenol as a standard) to determine the presence and amount ofalkaline phosphate activity. Protein concentrations are determined bythe Biorad method. Alkaline phosphatase activity is calculated inunits/mg protein, where 1 unit=1 nmol p-nitrophenol liberated/30 minutesat 37° C. OP-1 induces a five-fold increase in the specific activity ofalkaline phosphate by this method. Agonists are expected to have similarinduction effects. Antagonists should inhibit or otherwise interferewith OP1 binding, and diminished alkaline phophatase induction shouldresult when the assay is performed with an antagonist in the presence ofa limiting amount of OP1.

Induction of PTH-Mediated cAMP.

The effect of a morphogen analog on parathyroid hormone-mediated cAMPproduction in rat osteoblasts in vitro may be demonstrated as follows.

Rat osteoblasts are prepared and cultured in a multiwell plate asdescribed above. The cultured cells then are divided into three groups:(1) wells which receive, for example, 1.0, 10.0 and 40.0 ng OP-1/mlmedium); (2) wells which receive the candidate analog at variousconcentration ranges; and (3) a control group which receives noadditional factors. The plate is then incubated for another 72 hours. Atthe end of the 72 hours the cells are treated with medium containing0.5% bovine serum albumin (BSA) and 1 mM 3-isobutyl-1-methylxanthine for20 minutes followed by the addition into half of the wells of humanrecombinant parathyroid hormone (hPTH, Sigma, St. Louis) at aconcentration of 200 ng/ml for 10 minutes. The cell layer then isextracted from each well with 0.5 ml of 1% Triton X-100. The cAMP levelsthen are determined using a radioimmunoassay kit (e.g., Amersham,Arlington Heights, Illinois). OP-1 doubles cAMP production in thepresence of PTH. Agonists are expected to have similar inductioneffects. Antagonists are expected to inhibit or otherwise interfere withOP1 binding, and diminished cAMP production should result when the assayis performed with an antagonist in the presence of limiting the amountof OP1.

Induction of Osteocalcin Production

Osteocalcin is a bone-specific protein synthesized by osteoblasts whichplays an integral role in the rate of bone mineralization in vivo.Circulating levels of osteocalcin in serum are used as a marker forosteoblast activity and bone formation in vivo. Induction of osteocalcinsynthesis in osteoblast-enriched cultures can be used to demonstratemorphogenic efficacy in vitro.

Rat osteoblasts are prepared and cultured in a multi-well plate asabove. In this experiment the medium is supplemented with 10% FBS, andon day 2, cells are fed with fresh medium supplemented with fresh 10 mMb-glycerophosphate (Sigma, Inc.). Beginning on day 5 and twice weeklythereafter, cells are fed with a complete mineralization mediumcontaining all of the above components plus fresh L(+)-ascorbate, at afinal concentration of 50 mg/ml medium. OP1 or OP1 analog then is addedto the wells directly, e.g., in 50% acetonitrile (or 50% ethanol)containing 0.1% trifluoroacetic acid (TFA), at no more than 5 ml OP1/mlmedium. Control wells receive solvent vehicle only. The cells then arere-fed and the conditioned medium sample diluted 1:1 in standardradioimmunoassay buffer containing standard protease inhibitors andstored at -20° C. until assayed for osteocalcin. Osteocalcin synthesisis measured by standard radioimmunoassay using a commercially availableosteocalcin-specific antibody and can be confirmed by Northern blotanalysis to calculate the amount of osteocalcin mRNA produced in thepresence and absence of OP-1 or OP1 analog. OP-1 induces adose-dependent increase in osteocalcin production (5-fold increase using25 ng of OP-1 protein/ml), and a 20-fold increase in osteocalcin mRNA.Agonists are expected to have similar induction effects; antagonists areexpected to inhibit or otherwise interfere with OP1 binding, therebysubstantially interfering with osteocalcin induction in the presence ofa limiting amount of OP1.

Mineralization is determined on long term cultures (13 day) using amodified von Kossa staining technique on fixed cell layers: cells arefixed in fresh 4% paraformaldehyde at 23° C. for 10 min, followingrinsing cold 0.9% NaCl. Fixed cells then are stained for endogenousalkaline phosphatase at pH 9.5 for 10 min, using a commerciallyavailable kit (Sigma, Inc.) Purple stained cells then are dehydratedwith methanol and air dried. After 30 min incubation in 3% AgNO₃ in thedark, H₂ O-rinsed samples are exposed for 30 sec to 254 nm UV light todevelop the black silver-stained phosphate nodules. Individualmineralized foci (at least 20 mm in size) are counted under a dissectingmicroscope and expressed as nodules/culture. OP-1 induces a 20-foldincrease in initial mineralization rate. Agonists are expected to havesimilar induction effects; antagonists are expected to inhibit orotherwise interfere with OP1 binding, thereby inhibiting mineralizationinduction in the presence of a limiting amount of OP1.

9.2 Induction of a Constructed Reporter Gene

Alternatively, a reporter gene construct can be used to determine theability of candidate molecule to induce signal transduction across amembrane following receptor binding. For example, PAI-1 protein,(Plasminogen Activator Inhibitor-1) expression can be induced by OP-1 inMv1Lu- cells (see above). Also, as demontrated above, these cellsexpress ALK-2, -3 and -6 surface receptors. In addition, preliminarystudies indicate that ALK-1, when overexpressed in a chemicallymutagenized derivative of these cells, also apparently mediates PAI-1induction in the presence of OP1.

Accordingly, PAI-1 promoter elements can be fused to a reporter gene andinduction of the reporter gene monitored following incubation of thetransfected cell with a candidate analog. As one example, the luciferasereporter gene can be used, in, for example, the construct p3TP-Luxdescribed by Wrana et al. (1992) Cell 71: 1003-1014 and Attisano et al.(1993) Cell 74: 671-680. This reporter gene construct includes a regionof the human PAI-1 gene promoter in combination with three sets oftetradecanoyl phorbol--acetate responsive elements upstream of thelucifrase open reading frame.

In a typical assay, transfected cells starved in DMEM containing 0.1%fetal bovine serum and antibiotics (e.g., 100 units/ml penicillin and 50μg/ml streptomycin) for 6 hrs., and then exposed to ligand for 24 hr.Luciferase activity in the cell lysate then is measured using aluminometer in the luciferase assay system, according to themanufacturer's protocol(Promega). In MvlLu mutant cells, "R mutant"cells co-transfected with ALK-2 and Act RII, OP1 mediated induction ofluciferase activity.

9.3 Inhibition of Epithelial cell proliferation

OP1 is known to inhibit epithelial cells. Thus, the ability of acandidate analog to inhibit cell proliferation, as measured by ³H-thymidine uptake by an epithelial cell can be used in an assay toevaluate signal transduction activity of the candidate. Analogscompetent to inhibit epithelial cell growth are contemplated to haveparticular utility in therapeutic applications where limitation of aproliferating cell population is desired. Such applications includechemotherapies and radiation therapies where limiting the growth of anormally proliferating population of cells can protect these cells fromthe cytotoxic effects of these cancer therapies. (see e.g., W094/06420).In addition, psoriasis and other tissue disorders resulting fromuncontrolled cell proliferation, including benign and malignantneoplasties, can be modulated by use of an OP1 analog.

As an example, mink lung epithelial cell growth is inhibited by OP-1.(see, PCT US93/08885; W094/06420.) As described above, derivatives ofthese cells e.g., "R-4 mutants", clone 4-2, Laiho et al. (1990) J. Biol.Chem. 265: 18518-18524! can be transfected with DNA encodingOP1-specific receptors and induced to express these receptors. Thetransfected cells, then can be assayed for a candidate analog's abilityto block cell growth. As one example, when R-4 cells are transfectedwith ALK-3 under a Zn²⁺ -inducible promoter, and induced to express thereceptor following induction with ZnCl₂, cell growth can be inhibited inthe presence of OP1 in a dose dependent manner. Preliminary experimentswith ALK-1 indicates that this receptor also can mediate thisOP-1-specific effect.

In a typical assay, cells are seeded in 24-well cell culture plates at adensity of 10⁴ cells per well in DMEM with 10% FBS, and incubatedovernight. The medium is replaced with DMEM containing 0.2% FBS and 100uM ZnLC₂, and the cells are incubated for 5 h, after which the medium isreplaced with fresh DMEM containing 0.2% FBS, 100 uM ZnCL₂ and variousconcentrations of OP-1 or an analog candidate. After 16 h of incubation,0.25 ci of ³ H!thymidine (Amersham) are added and the cells incubatedfor an additional 2 h. Thereafter, the cells are fixed in 10%trichloroacetic acid for more than 15 min on ice, and solubilized with1M NaOH. The cell extracts are neutralized with 1M HCl and ³ Hradioactivity determined in a liquid scintillation counter.

EXAMPLE 10 Screening Assay for Compounds Which Alter Engogenous OP1Receptor Expression Levels

Candidate compound(s) which can be administered to affect the level of agiven endogenous OP1 receptor can be found using the following screeningassay, in which the level of OP1 receptor production by a cell typewhich produces measurable levels of the receptor is determined byincubating the cell in culture with and without the candidate compound,in order to assess the effects of the compound on the cell. This alsocan be accomplished by detection of the OP1 receptor either at theprotein level by Western blot or immunolocalization, or at the RNA levelby Northern blot or in situ hydridization. The protocol is based on aprocedure for identifying compounds which alter endogenous levels of OP1expression, a detailed description also may be found in PCT US 92/07359,incorporated herein by reference.

Cell cultures of, for example, bone, brain, intestine, lung, heart, eye,breast, gonads, kidney, adrenals, urinary bladder, brain, or otherorgans, may be prepared as described widely in the literature. Forexample, kidneys may be explanted from neonatal or new born or young oradult rodents (mouse or rat) and used in organ culture as whole orsliced (1-4 mm) tissues. Primary tissue cultures and established celllines, also derived from kidney, adrenals, urinary, bladder, brain,mammary, or other tissues may be established in multiwell plates (6 wellor 24 well) according to conventional cell culture techniques, and arecultured in the absence or presence of serum for a period of time (1-7days). Cells can be cultured, for example, in Dulbecco's Modified Eaglemedium (Gibco, Long Island, N.Y.) containing serum (e.g., fetal calfserum at 1%-10%, Gibco) or in serum-deprived medium, as desired, or indefined medium (e.g., containing insulin, transferrin, glucose, albumin,or other growth factors).

Cell samples for testing the level of OP1 receptor production arecollected periodically and evaluated for receptor production byimmunoblot analysis (Sambrook et al., eds., 1989, Molecular Cloning,Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), or, alternatively,a portion of the cell culture itself can be collected periodically andused to prepare polyA+ RNA for mRNA analysis by Northern blot analysis.To monitor de novo receptor synthesis, some cultures are labeledaccording to conventional procedures with an ³⁵ S-methionine/³⁵S-cysteine mixture for 6-24 hours and then evaluated to quantitatereceptor synthesis by conventional immunoassay methods. Alternatively,anti-receptor antibodies may be labelled and incubated with the cells orcell lysates, and the bound complexes detected and quantitated byconventional means, such as those described hereinabove. Northern blotsmay be performed using a portion of the OP1 receptor coding sequence tocreate hybridization probes, and following the RNA hybridizationprotocol described herein.

EXAMPLE 11 General Formulation/Adninistration Considerations

The analogs and constructs described herein can be provided to anindividual as part of a therapy to enhance, inhibit, or otherwisemodulate the in vivo binding interaction between OP1 and one or moreOP1-specific cell surface receptors. The molecules then comprise part ofa pharmaceutical composition as described herein below and can beadministered by any suitable means, preferably directly or systemically,e.g., parenterally or orally. Where the therapeutic molecule is to beprovided directly (e.g., locally, as by injection, to a desired tissuesite), or parenterally, such as by intravenous, subcutaneous,intramuscular, intraorbital, ophthalmic, intraventricular, intracranial,intracapsular, intraspinal, intracisternal, intraperitoneal, buccal,rectal, vaginal, intranasal or by aerosol administration, thetherapeutic preferably comprises part of an aqueous solution. Thesolution preferably is physiologically acceptable so that in addition todelivery of the desired morphogen to the patient, the solution does nototherwise adversely affect the patient's electrolyte and volume balance.The aqueous medium for the therapeutic molecule thus may comprise normalphysiologic saline (0.9% NaCl, 0.15M), pH 7-7.4 or otherpharmaceutically acceptable salts thereof.

Useful solutions for oral or parenteral administration can be preparedby any of the methods well known in the pharmaceutical art, described,for example, in Remington's Pharmaceutical Sciences, (Gennaro, A., ed.),Mack Pub., 1990. Formulations may include, for example, polyalkyleneglycols such as polyethylene glycol, oils of vegetable origin,hydrogenated naphthalenes, and the like. Formulations for directadministration, in particular, can include glycerol and othercompositions of high viscosity. Biocompatible, preferably bioresorbablepolymers, including, for example, hyaluronic acid, collagen, tricalciumphosphate, polybutyrate, polylactide, polyglycolide andlactide/glycolide copolymers, may be useful excipients to control therelease of the morphogen in vivo.

Other potentially useful parenteral delivery systems for thesetherapeutic molecules include ethylene-vinyl acetate copolymerparticles, osmotic pumps, implantable infusion systems, and liposomes.Formulations for inhalation administration may contain as excipients,for example, lactose, or can be aqueous solutions containing, forexample, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate,or oily solutions for administration in the form of nasal drops, or as agel to be applied intranasally.

Alternatively, the morphogens described herein may be administeredorally.

The therapeutic molecules also can be associated with means fortargeting the therapeutic to a desired tissue. For example, tetracyclineand diphosphonates (bisphosphonates) are known to bind to bone mineral,particularly at zones of bone remodeling, when they are providedsystemically in a mammal. Accordingly, these molecules may be includedas useful agents for targeting therapeutics to bone tissue.Alternatively, an antibody or other binding protein that interactsspecifically with a surface molecule on the desired target tissue cellsalso can be used. Such targeting molecules further can be covalentlyassociated to the therapeutic molecule e.g., by chemical crosslinking,or by using standard genetic engineering means to create, for example,an acid labile bond such as an Asp-Pro linkage. Useful targetingmolecules can be designed, for example, using the single chain bindingsite technology disclosed, for example, in U.S. Pat. No. 5,091,513.

Finally, therapeutic molecules can be administered alone or incombination with other molecules known to have a beneficial effect ontissue morphogenesis, including molecules capable of tissue repair andregeneration and/or inhibiting inflammation. Examples of usefulcofactors for stimulating bone tissue growth in osteoporoticindividuals, for example, include but are not limited to, vitamin D₃,calcitonin, prostaglandins, parathyroid hormone, dexamethasone, estrogenand IGF-I or IGF-II. Useful cofactors for nerve tissue repair andregeneration can include nerve growth factors. Other useful cofactorsinclude symptom-alleviating cofactors, including antiseptics,antibiotics, antiviral and antifungal agents and analgesics andanesthetics.

Therapeutic molecules further can be formulated into pharmaceuticalcompositions by admixture with pharmaceutically acceptable nontoxicexcipients and carriers. As noted above, such compositions can beprepared for parenteral administration, particularly in the form ofliquid solutions or suspensions; for oral administration, particularlyin the form of tablets or capsules; or intranasally, particularly in theform of powders, nasal drops or aerosols. Where adhesion to a tissuesurface is desired the composition may include the morphogen dispersedin a fibrinogen-thrombin composition or other bioadhesive such as isdisclosed, for example in PCT US91/09275, the disclosure of which isincorporated herein by reference. The composition then can be painted,sprayed or otherwise applied to the desired tissue surface.

The compositions can be formulated for parenteral or oral administrationto humans or other mammals in therapeutically effective amounts, e.g.,amounts which provide appropriate concentrations of the analog to targettissue for a time sufficient to induce the desired effect.

Where the analog is to be used as part of a transplant procedure, it canbe provided to the living tissue or organ to be transplanted prior toremoval of tissue or organ from the donor. The analog may be provided tothe donor host directly, as by injection of a formulation comprising theanalog into the tissue, or indirectly, e.g., by oral or parenteraladministration, using any of the means described above.

Alternatively or, in addition, once removed from the donor, the organ orliving tissue can be placed in a preservation solution containing thetherapeutic molecule. In addition, the recipient also preferably isprovided with the analog just prior to, or concomitant with,transplantation. In all cases, the analog can be administered directlyto the tissue at risk, as by injection to the tissue, or it may beprovided systemically, either by oral or parenteral administration,using any of the methods and formulations described herein and/or knownin the art.

Where the therapeutic molecule comprises part of a tissue or organpreservation solution, any commercially available preservation solutioncan be used to advantage. For example, useful solutions known in the artinclude Collins solution, Wisconsin solution, Belzer solution,Eurocollins solution and lactated Ringer's solution. Generally, an organpreservation solution usually possesses one or more of the followingproperties: (a) an osmotic pressure substantially equal to that of theinside of a mammalian cell,(solutions typically are hyperosmolar andhave K+ and/or Mg++ ions present in an amount sufficient to produce anosmotic pressure slightly higher than the inside of a mammalian cell);(b) the solution typically is capable of maintaining substantiallynormal ATP levels in the cells; and (c) the solution usually allowsoptimum maintenance of glucose metabolism in the cells. Organpreservation solutions also may contain anticoagulants, energy sourcessuch as glucose, fructose and other sugars, metabolites, heavy metalchelators, glycerol and other materials of high viscosity to enhancesurvival at low temperatures, free oxygen radical inhibiting and/orscavenging agents and a pH indicator. A detailed description ofpreservation solutions and useful components can be found, for example,in U.S. Pat. No. 5,002,965, the disclosure of which is incorporatedherein by reference.

As will be appreciated by those skilled in the art, the concentration ofthe compounds described in a therapeutic composition will vary dependingupon a number of factors, including the dosage of the drug to beadministered, the chemical characteristics (e.g., hydrophobicity) of thecompounds employed, and the route of administration. The preferreddosage of drug to be administered also is likely to depend on suchvariables as the type and extent of tissue loss or defect, the overallhealth status of the particular patient, the relative biologicalefficacy of the compound selected, the formulation of the compound, thepresence and types of excipients in the formulation, and the route ofadministration. In general terms, the therapeutic molecules of thisinvention may be provided to and individual where typical dose rangesare from about 10 ng/kg to about 1 g/kg of body weight per day; apreferred dose range being from about 0.1 mg/kg to 100 mg/kg of bodyweight. No obvious morphogen-induced pathological lesions are inducedwhen mature morphogen (e.g., OP-1, 20 mg) is administered daily tonormal growing rats for 21 consecutive days. Moreover, 10 mg systemicinjections of morphogen (e.g., OP-1) injected daily for 10 days intonormal newborn mice does not produce any gross abnormalities.

Other Embodiments

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 18    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1509 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..1509    (D) OTHER INFORMATION: /product="Human ALK1"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    ATGACCTTGGGCTCCCCCAGGAAAGGCCTTCTGATGCTGCTGATGGCC48    MetThrLeuGlySerProArgLysGlyLeuLeuMetLeuLeuMetAla    151015    TTGGTGACCCAGGGAGACCCTGTGAAGCCGTCTCGGGGCCCGCTGGTG96    LeuValThrGlnGlyAspProValLysProSerArgGlyProLeuVal    202530    ACCTGCACGTGTGAGAGCCCACATTGCAAGGGGCCTACCTGCCGGGGG144    ThrCysThrCysGluSerProHisCysLysGlyProThrCysArgGly    354045    GCCTGGTGCACAGTAGTGCTGGTGCGGGAGGAGGGGAGGCACCCCCAG192    AlaTrpCysThrValValLeuValArgGluGluGlyArgHisProGln    505560    GAACATCGGGGCTGCGGGAACTTGCACAGGGAGCTCTGCAGGGGGCGC240    GluHisArgGlyCysGlyAsnLeuHisArgGluLeuCysArgGlyArg    65707580    CCCACCGAGTTCGTCAACCACTACTGCTGCGACAGCCACCTCTGCAAC288    ProThrGluPheValAsnHisTyrCysCysAspSerHisLeuCysAsn    859095    CACAACGTGTCCCTGGTGCTGGAGGCCACCCAACCTCCTTCGGAGCAG336    HisAsnValSerLeuValLeuGluAlaThrGlnProProSerGluGln    100105110    CCGGGAACAGATGGCCAGCTGGCCCTGATCCTGGGCCCCGTGCTGGCC384    ProGlyThrAspGlyGlnLeuAlaLeuIleLeuGlyProValLeuAla    115120125    TTGCTGGCCCTGGTGGCCCTGGGTGTCCTGGGCCTGTGGCATGTCCGA432    LeuLeuAlaLeuValAlaLeuGlyValLeuGlyLeuTrpHisValArg    130135140    CGGAGGCAGGAGAAGCAGCGTGGCCTGCACAGCGAGCTGGGAGAGTCC480    ArgArgGlnGluLysGlnArgGlyLeuHisSerGluLeuGlyGluSer    145150155160    AGTCTCATCCTGAAAGCATCTGAGCAGGGCGACACGATGTTGGGGGAC528    SerLeuIleLeuLysAlaSerGluGlnGlyAspThrMetLeuGlyAsp    165170175    CTCCTGGACAGTGACTGCACCACAGGGAGTGGCTCAGGGCTCCCCTTC576    LeuLeuAspSerAspCysThrThrGlySerGlySerGlyLeuProPhe    180185190    CTGGTGCAGAGGACAGTGGCACGGCAGGTTGCCTTGGTGGAGTGTGTG624    LeuValGlnArgThrValAlaArgGlnValAlaLeuValGluCysVal    195200205    GGAAAAGGCCGCTATGGCGAAGTGTGGCGGGGCTTGTGGCACGGTGAG672    GlyLysGlyArgTyrGlyGluValTrpArgGlyLeuTrpHisGlyGlu    210215220    AGTGTGGCCGTCAAGATCTTCTCCTCGAGGGATGAACAGTCCTGGTTC720    SerValAlaValLysIlePheSerSerArgAspGluGlnSerTrpPhe    225230235240    CGGGAGACTGAGATCTATAACACAGTATTGCTCAGACACGACAACATC768    ArgGluThrGluIleTyrAsnThrValLeuLeuArgHisAspAsnIle    245250255    CTAGGCTTCATCGCCTCAGACATGACCTCCCGCAACTCGAGCACGCAG816    LeuGlyPheIleAlaSerAspMetThrSerArgAsnSerSerThrGln    260265270    CTGTGGCTCATCACGCACTACCACGAGCACGGCTCCCTCTACGACTTT864    LeuTrpLeuIleThrHisTyrHisGluHisGlySerLeuTyrAspPhe    275280285    CTGCAGAGACAGACGCTGGAGCCCCATCTGGCTCTGAGGCTAGCTGTG912    LeuGlnArgGlnThrLeuGluProHisLeuAlaLeuArgLeuAlaVal    290295300    TCCGCGGCATGCGGCCTGGCGCACCTGCACGTGGAGATCTTCGGTACA960    SerAlaAlaCysGlyLeuAlaHisLeuHisValGluIlePheGlyThr    305310315320    CAGGGCAAACCAGCCATTGCCCACCGCGACTTCAAGAGCCGCAATGTG1008    GlnGlyLysProAlaIleAlaHisArgAspPheLysSerArgAsnVal    325330335    CTGGTCAAGAGCAACCTGCAGTGTTGCATCGCCGACCTGGGCCTGGCT1056    LeuValLysSerAsnLeuGlnCysCysIleAlaAspLeuGlyLeuAla    340345350    GTGATGCACTCACAGGGCAGCGATTACCTGGACATCGGCAACAACCCG1104    ValMetHisSerGlnGlySerAspTyrLeuAspIleGlyAsnAsnPro    355360365    AGAGTGGGCACCAAGCGGTACATGGCACCCGAGGTGCTGGACGAGCAG1152    ArgValGlyThrLysArgTyrMetAlaProGluValLeuAspGluGln    370375380    ATCCGCACGGACTGCTTTGAGTCCTACAAGTGGACTGACATCTGGGCC1200    IleArgThrAspCysPheGluSerTyrLysTrpThrAspIleTrpAla    385390395400    TTTGGCCTGGTGCTGTGGGAGATTGCCCGCCGGACCATCGTGAATGGC1248    PheGlyLeuValLeuTrpGluIleAlaArgArgThrIleValAsnGly    405410415    ATCGTGGAGGACTATAGACCACCCTTCTATGATGTGGTGCCCAATGAC1296    IleValGluAspTyrArgProProPheTyrAspValValProAsnAsp    420425430    CCCAGCTTTGAGGACATGAAGAAGGTGGTGTGTGTGGATCAGCAGACC1344    ProSerPheGluAspMetLysLysValValCysValAspGlnGlnThr    435440445    CCCACCATCCCTAACCGGCTGGCTGCAGACCCGGTCCTCTCAGGCCTA1392    ProThrIleProAsnArgLeuAlaAlaAspProValLeuSerGlyLeu    450455460    GCTCAGATGATGCGGGAGTGCTGGTACCCAAACCCCTCTGCCCGACTC1440    AlaGlnMetMetArgGluCysTrpTyrProAsnProSerAlaArgLeu    465470475480    ACCGCGCTGCGGATCAAGAAGACACTACAAAAAATTAGCAACAGTCCA1488    ThrAlaLeuArgIleLysLysThrLeuGlnLysIleSerAsnSerPro    485490495    GAGAAGCCTAAAGTGATTCAA1509    GluLysProLysValIleGln    500    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 503 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetThrLeuGlySerProArgLysGlyLeuLeuMetLeuLeuMetAla    151015    LeuValThrGlnGlyAspProValLysProSerArgGlyProLeuVal    202530    ThrCysThrCysGluSerProHisCysLysGlyProThrCysArgGly    354045    AlaTrpCysThrValValLeuValArgGluGluGlyArgHisProGln    505560    GluHisArgGlyCysGlyAsnLeuHisArgGluLeuCysArgGlyArg    65707580    ProThrGluPheValAsnHisTyrCysCysAspSerHisLeuCysAsn    859095    HisAsnValSerLeuValLeuGluAlaThrGlnProProSerGluGln    100105110    ProGlyThrAspGlyGlnLeuAlaLeuIleLeuGlyProValLeuAla    115120125    LeuLeuAlaLeuValAlaLeuGlyValLeuGlyLeuTrpHisValArg    130135140    ArgArgGlnGluLysGlnArgGlyLeuHisSerGluLeuGlyGluSer    145150155160    SerLeuIleLeuLysAlaSerGluGlnGlyAspThrMetLeuGlyAsp    165170175    LeuLeuAspSerAspCysThrThrGlySerGlySerGlyLeuProPhe    180185190    LeuValGlnArgThrValAlaArgGlnValAlaLeuValGluCysVal    195200205    GlyLysGlyArgTyrGlyGluValTrpArgGlyLeuTrpHisGlyGlu    210215220    SerValAlaValLysIlePheSerSerArgAspGluGlnSerTrpPhe    225230235240    ArgGluThrGluIleTyrAsnThrValLeuLeuArgHisAspAsnIle    245250255    LeuGlyPheIleAlaSerAspMetThrSerArgAsnSerSerThrGln    260265270    LeuTrpLeuIleThrHisTyrHisGluHisGlySerLeuTyrAspPhe    275280285    LeuGlnArgGlnThrLeuGluProHisLeuAlaLeuArgLeuAlaVal    290295300    SerAlaAlaCysGlyLeuAlaHisLeuHisValGluIlePheGlyThr    305310315320    GlnGlyLysProAlaIleAlaHisArgAspPheLysSerArgAsnVal    325330335    LeuValLysSerAsnLeuGlnCysCysIleAlaAspLeuGlyLeuAla    340345350    ValMetHisSerGlnGlySerAspTyrLeuAspIleGlyAsnAsnPro    355360365    ArgValGlyThrLysArgTyrMetAlaProGluValLeuAspGluGln    370375380    IleArgThrAspCysPheGluSerTyrLysTrpThrAspIleTrpAla    385390395400    PheGlyLeuValLeuTrpGluIleAlaArgArgThrIleValAsnGly    405410415    IleValGluAspTyrArgProProPheTyrAspValValProAsnAsp    420425430    ProSerPheGluAspMetLysLysValValCysValAspGlnGlnThr    435440445    ProThrIleProAsnArgLeuAlaAlaAspProValLeuSerGlyLeu    450455460    AlaGlnMetMetArgGluCysTrpTyrProAsnProSerAlaArgLeu    465470475480    ThrAlaLeuArgIleLysLysThrLeuGlnLysIleSerAsnSerPro    485490495    GluLysProLysValIleGln    500    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2724 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 104..1630    (D) OTHER INFORMATION: /product="Human ALK2"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    CTCCGAGTACCCCAGTGACCAGAGTGAGAGAAGCTCTGAACGAGGGCACGCGGCTTGAAG60    GACTGTGGGCAGATGTGACCAAGAGCCTGCATTAAGTTGTACAATGGTAGATGGA115    MetValAspGly    GTGATGATTCTTCCTGTGCTTATCATGATTGCTCTCCCCTCCCCTAGT163    ValMetIleLeuProValLeuIleMetIleAlaLeuProSerProSer    5101520    ATGGAAGATGAGAAGCCCAAGGTCAACCCCAAACTCTACATGTGTGTG211    MetGluAspGluLysProLysValAsnProLysLeuTyrMetCysVal    253035    TGTGAAGGTCTCTCCTGCGGTAATGAGGACCACTGTGAAGGCCAGCAG259    CysGluGlyLeuSerCysGlyAsnGluAspHisCysGluGlyGlnGln    404550    TGCTTTTCCTCACTGAGCATCAACGATGGCTTCCACGTCTACCAGAAA307    CysPheSerSerLeuSerIleAsnAspGlyPheHisValTyrGlnLys    556065    GGCTGCTTCCAGGTTTATGAGCAGGGAAAGATGACCTGTAAGACCCCG355    GlyCysPheGlnValTyrGluGlnGlyLysMetThrCysLysThrPro    707580    CCGTCCCCTGGCCAAGCTGTGGAGTGCTGCCAAGGGGACTGGTGTAAC403    ProSerProGlyGlnAlaValGluCysCysGlnGlyAspTrpCysAsn    859095100    AGGAACATCACGGCCCAGCTGCCCACTAAAGGAAAATCCTTCCCTGGA451    ArgAsnIleThrAlaGlnLeuProThrLysGlyLysSerPheProGly    105110115    ACACAGAATTTCCACTTGGAGGTTGGCCTCATTATTCTCTCTGTAGTG499    ThrGlnAsnPheHisLeuGluValGlyLeuIleIleLeuSerValVal    120125130    TTCGCAGTATGTCTTTTAGCCTGCCTGCTGGGAGTTGCTCTCCGAAAA547    PheAlaValCysLeuLeuAlaCysLeuLeuGlyValAlaLeuArgLys    135140145    TTTAAAAGGCGCAACCAAGAACGCCTCAATCCCCGAGACGTGGAGTAT595    PheLysArgArgAsnGlnGluArgLeuAsnProArgAspValGluTyr    150155160    GGCACTATCGAAGGGCTCATCACCACCAATGTTGGAGACAGCACTTTA643    GlyThrIleGluGlyLeuIleThrThrAsnValGlyAspSerThrLeu    165170175180    GCAGATTTATTGGATCATTCGTGTACATCAGGAAGTGGCTCTGGTCTT691    AlaAspLeuLeuAspHisSerCysThrSerGlySerGlySerGlyLeu    185190195    CCTTTTCTGGTACAAAGAACAGTGGCTCGCCAGATTACACTGTTGGAG739    ProPheLeuValGlnArgThrValAlaArgGlnIleThrLeuLeuGlu    200205210    TGTGTCGGGAAAGGCAGGTATGGTGAGGTGTGGAGGGGCAGCTGGCAA787    CysValGlyLysGlyArgTyrGlyGluValTrpArgGlySerTrpGln    215220225    GGGGAAAATGTTGCCGTGAAGATCTTCTCCTCCCGTGATGAGAAGTCA835    GlyGluAsnValAlaValLysIlePheSerSerArgAspGluLysSer    230235240    TGGTTCAGGGAAACGGAATTGTACAACACTGTGATGCTGAGGCATGAA883    TrpPheArgGluThrGluLeuTyrAsnThrValMetLeuArgHisGlu    245250255260    AATATCTTAGGTTTCATTGCTTCAGACATGACATCAAGACACTCCAGT931    AsnIleLeuGlyPheIleAlaSerAspMetThrSerArgHisSerSer    265270275    ACCCAGCTGTGGTTAATTACACATTATCATGAAATGGGATCGTTGTAC979    ThrGlnLeuTrpLeuIleThrHisTyrHisGluMetGlySerLeuTyr    280285290    GACTATCTTCAGCTTACTACTCTGGATACAGTTAGCTGCCTTCGAATA1027    AspTyrLeuGlnLeuThrThrLeuAspThrValSerCysLeuArgIle    295300305    GTGCTGTCCATAGCTAGTGGTCTTGCACATTTGCACATAGAGATATTT1075    ValLeuSerIleAlaSerGlyLeuAlaHisLeuHisIleGluIlePhe    310315320    GGGACCCAAGGGAAACCAGCCATTGCCCATCGAGATTTAAAGAGCAAA1123    GlyThrGlnGlyLysProAlaIleAlaHisArgAspLeuLysSerLys    325330335340    AATATTCTGGTTAAGAAGAATGGACAGTGTTGCATAGCAGATTTGGGC1171    AsnIleLeuValLysLysAsnGlyGlnCysCysIleAlaAspLeuGly    345350355    CTGGCAGTCATGCATTCCCAGAGCACCAATCAGCTTGATGTGGGGAAC1219    LeuAlaValMetHisSerGlnSerThrAsnGlnLeuAspValGlyAsn    360365370    AATCCCCGTGTGGGCACCAAGCGCTACATGGCCCCCGAAGTTCTAGAT1267    AsnProArgValGlyThrLysArgTyrMetAlaProGluValLeuAsp    375380385    GAAACCATCCAGGTGGATTGTTTCGATTCTTATAAAAGGGTCGATATT1315    GluThrIleGlnValAspCysPheAspSerTyrLysArgValAspIle    390395400    TGGGCCTTTGGACTTGTTTTGTGGGAAGTGGCCAGGCGGATGGTGAGC1363    TrpAlaPheGlyLeuValLeuTrpGluValAlaArgArgMetValSer    405410415420    AATGGTATAGTGGAGGATTACAAGCCACCGTTCTACGATGTGGTTCCC1411    AsnGlyIleValGluAspTyrLysProProPheTyrAspValValPro    425430435    AATGACCCAAGTTTTGAAGATATGAGGAAGGTAGTCTGTGTGGATCAA1459    AsnAspProSerPheGluAspMetArgLysValValCysValAspGln    440445450    CAAAGGCCAAACATACCCAACAGATGGTTCTCAGACCCGACATTAACC1507    GlnArgProAsnIleProAsnArgTrpPheSerAspProThrLeuThr    455460465    TCTCTGGCCAAGCTAATGAAAGAATGCTGGTATCAAAATCCATCCGCA1555    SerLeuAlaLysLeuMetLysGluCysTrpTyrGlnAsnProSerAla    470475480    AGACTCACAGCACTGCGTATCAAAAAGACTTTGACCAAAATTGATAAT1603    ArgLeuThrAlaLeuArgIleLysLysThrLeuThrLysIleAspAsn    485490495500    TCCCTCGACAAATTGAAAACTGACTGTTGACATTTTCATAGTGTCAA1650    SerLeuAspLysLeuLysThrAspCys    505    GAAGGAAGATTTGACGTTGTTGTCATTGTCCAGCTGGGACCTAATGCTGGCCTGACTGGT1710    TGTCAGAATGGAATCCATCTGTCTCCCTCCCCAAATGGCTGCTTTGACAAGGCAGACGTC1770    GTACCCAGCCATGTGTTGGGGAGACATCAAAACCACCCTAACCTCGCTCGATGACTGTGA1830    ACTGGGCATTTCACGAACTGTTCACACTGCAGAGACTAATGTTGGACAGACACTGTTGCA1890    AAGGTAGGGACTGGAGGAACACAGAGAAATCCTAAAAGAGATCTGGGCATTAAGTCAGTG1950    GCTTTGCATAGCTTTCACAAGTCTCCTAGACACTCCCCACGGGAAACTCAAGGAGGTGGT2010    GAATTTTTAATCAGCAATATTGCCTGTGCTTCTCTTCTTTATTGCACTAGGAATTCTTTG2070    CATTCCTTACTTGCACTGTTACTCTTAATTTTAAAGACCCAACTTGCCAAAATGTTGGCT2130    GCGTACTCCACTGGTCTGTCTTTGGATAATAGGAATTCAATTTGGCAAAACAAAATGTAA2190    TGTCAGACTTTGCTGCATTTTACACATGTGCTGATGTTTACAATGATGCCGAACATTAGG2250    AATTGTTTATACACAACTTTGCAAATTATTTATTACTTGTGCACTTAGTAGTTTTTACAA2310    AACTGCTTTGTGCATATGTTAAAGCTTATTTTTATGTGGTCTTATGATTTTATTACAGAA2370    ATGTTTTTAACACTATACTCTAAAATGGACATTTTCTTTTATTATCAGTTAAAATCACAT2430    TTTAAGTGCTTCACATTTGTATGTGTGTAGACTGTAACTTTTTTTCAGTTCATATGCAGA2490    ACGTATTTAGCCATTACCCACGTGACACCACCGAATATATTATCGATTTAGAAGCAAAGA2550    TTTCAGTAGAATTTTAGTCCTGAACGCTACGGGGAAAATGCATTTTCTTCAGAATTATCC2610    ATTACGTGCATTTAAACTCTGCCAGAAAAAAATAACTATTTTGTTTTAATCTACTTTTTG2670    TATTTAGTAGTTATTTGTATAAATTAAATAAACTGTTTTCAAGTCAAAAAAAAA2724    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 509 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetValAspGlyValMetIleLeuProValLeuIleMetIleAlaLeu    151015    ProSerProSerMetGluAspGluLysProLysValAsnProLysLeu    202530    TyrMetCysValCysGluGlyLeuSerCysGlyAsnGluAspHisCys    354045    GluGlyGlnGlnCysPheSerSerLeuSerIleAsnAspGlyPheHis    505560    ValTyrGlnLysGlyCysPheGlnValTyrGluGlnGlyLysMetThr    65707580    CysLysThrProProSerProGlyGlnAlaValGluCysCysGlnGly    859095    AspTrpCysAsnArgAsnIleThrAlaGlnLeuProThrLysGlyLys    100105110    SerPheProGlyThrGlnAsnPheHisLeuGluValGlyLeuIleIle    115120125    LeuSerValValPheAlaValCysLeuLeuAlaCysLeuLeuGlyVal    130135140    AlaLeuArgLysPheLysArgArgAsnGlnGluArgLeuAsnProArg    145150155160    AspValGluTyrGlyThrIleGluGlyLeuIleThrThrAsnValGly    165170175    AspSerThrLeuAlaAspLeuLeuAspHisSerCysThrSerGlySer    180185190    GlySerGlyLeuProPheLeuValGlnArgThrValAlaArgGlnIle    195200205    ThrLeuLeuGluCysValGlyLysGlyArgTyrGlyGluValTrpArg    210215220    GlySerTrpGlnGlyGluAsnValAlaValLysIlePheSerSerArg    225230235240    AspGluLysSerTrpPheArgGluThrGluLeuTyrAsnThrValMet    245250255    LeuArgHisGluAsnIleLeuGlyPheIleAlaSerAspMetThrSer    260265270    ArgHisSerSerThrGlnLeuTrpLeuIleThrHisTyrHisGluMet    275280285    GlySerLeuTyrAspTyrLeuGlnLeuThrThrLeuAspThrValSer    290295300    CysLeuArgIleValLeuSerIleAlaSerGlyLeuAlaHisLeuHis    305310315320    IleGluIlePheGlyThrGlnGlyLysProAlaIleAlaHisArgAsp    325330335    LeuLysSerLysAsnIleLeuValLysLysAsnGlyGlnCysCysIle    340345350    AlaAspLeuGlyLeuAlaValMetHisSerGlnSerThrAsnGlnLeu    355360365    AspValGlyAsnAsnProArgValGlyThrLysArgTyrMetAlaPro    370375380    GluValLeuAspGluThrIleGlnValAspCysPheAspSerTyrLys    385390395400    ArgValAspIleTrpAlaPheGlyLeuValLeuTrpGluValAlaArg    405410415    ArgMetValSerAsnGlyIleValGluAspTyrLysProProPheTyr    420425430    AspValValProAsnAspProSerPheGluAspMetArgLysValVal    435440445    CysValAspGlnGlnArgProAsnIleProAsnArgTrpPheSerAsp    450455460    ProThrLeuThrSerLeuAlaLysLeuMetLysGluCysTrpTyrGln    465470475480    AsnProSerAlaArgLeuThrAlaLeuArgIleLysLysThrLeuThr    485490495    LysIleAspAsnSerLeuAspLysLeuLysThrAspCys    500505    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2932 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 310..1905    (D) OTHER INFORMATION: /product="Human ALK3"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GCTCCGCGCCGAGGGCTGGAGGATGCGTTCCCTGGGGTCCGGACTTATGAAAATATGCAT60    CAGTTTAATACTGTCTTGGAATTCATGAGATGGAAGCATAGGTCAAAGCTGTTTGGAGAA120    AATCAGAAGTACAGTTTTATCTAGCCACATCTTGGAGGAGTCGTAAGAAAGCAGTGGGAG180    TTGAAGTCATTGTCAAGTGCTTGCGATCTTTTACAAGAAAATCTCACTGAATGATAGTCA240    TTTAAATTGGTGAAGTAGCAAGACCAATTATTAAAGGTGACAGTACACAGGAAACATTAC300    AATTGAACAATGACTCAGCTATACATTTACATCAGATTATTGGGAGCC348    MetThrGlnLeuTyrIleTyrIleArgLeuLeuGlyAla    1510    TATTTGTTCATCATTTCTCGTGTTCAAGGACAGAATCTGGATAGTATG396    TyrLeuPheIleIleSerArgValGlnGlyGlnAsnLeuAspSerMet    152025    CTTCATGGCACTGGGATGAAATCAGACTCCGACCAGAAAAAGTCAGAA444    LeuHisGlyThrGlyMetLysSerAspSerAspGlnLysLysSerGlu    30354045    AATGGAGTAACCTTAGCACCAGAGGATACCTTGCCTTTTTTAAAGTGC492    AsnGlyValThrLeuAlaProGluAspThrLeuProPheLeuLysCys    505560    TATTGCTCAGGGCACTGTCCAGATGATGCTATTAATAACACATGCATA540    TyrCysSerGlyHisCysProAspAspAlaIleAsnAsnThrCysIle    657075    ACTAATGGACATTGCTTTGCCATCATAGAAGAAGATGACCAGGGAGAA588    ThrAsnGlyHisCysPheAlaIleIleGluGluAspAspGlnGlyGlu    808590    ACCACATTAGCTTCAGGGTGTATGAAATATGAAGGATCTGATTTTCAG636    ThrThrLeuAlaSerGlyCysMetLysTyrGluGlySerAspPheGln    95100105    TGCAAAGATTCTCCAAAAGCCCAGCTACGCCGGACAATAGAATGTTGT684    CysLysAspSerProLysAlaGlnLeuArgArgThrIleGluCysCys    110115120125    CGGACCAATTTATGTAACCAGTATTTGCAACCCACACTGCCCCCTGTT732    ArgThrAsnLeuCysAsnGlnTyrLeuGlnProThrLeuProProVal    130135140    GTCATAGGTCCGTTTTTTGATGGCAGCATTCGATGGCTGGTTTTGCTC780    ValIleGlyProPhePheAspGlySerIleArgTrpLeuValLeuLeu    145150155    ATTTCTATGGCTGTCTGCATAATTGCTATGATCATCTTCTCCAGCTGC828    IleSerMetAlaValCysIleIleAlaMetIleIlePheSerSerCys    160165170    TTTTGTTACAAACATTATTGCAAGAGCATCTCAAGCAGACGTCGTTAC876    PheCysTyrLysHisTyrCysLysSerIleSerSerArgArgArgTyr    175180185    AATCGTGATTTGGAACAGGATGAAGCATTTATTCCAGTTGGAGAATCA924    AsnArgAspLeuGluGlnAspGluAlaPheIleProValGlyGluSer    190195200205    CTAAAAGACCTTATTGACCAGTCACAAAGTTCTGGTAGTGGGTCTGGA972    LeuLysAspLeuIleAspGlnSerGlnSerSerGlySerGlySerGly    210215220    CTACCTTTATTGGTTCAGCGAACTATTGCCAAACAGATTCAGATGGTC1020    LeuProLeuLeuValGlnArgThrIleAlaLysGlnIleGlnMetVal    225230235    CGGCAAGTTGGTAAAGGCCGATATGGAGAAGTATGGATGGGCAAATGG1068    ArgGlnValGlyLysGlyArgTyrGlyGluValTrpMetGlyLysTrp    240245250    CGTGGCGAAAAAGTGGCGGTGAAAGTATTCTTTACCACTGAAGAAGCC1116    ArgGlyGluLysValAlaValLysValPhePheThrThrGluGluAla    255260265    AGCTGGTTTCGAGAAACAGAAATCTACCAAACTGTGCTAATGCGCCAT1164    SerTrpPheArgGluThrGluIleTyrGlnThrValLeuMetArgHis    270275280285    GAAAACATACTTGGTTTCATAGCGGCAGACATTAAAGGTACAGGTTCC1212    GluAsnIleLeuGlyPheIleAlaAlaAspIleLysGlyThrGlySer    290295300    TGGACTCAGCTCTATTTGATTACTGATTACCATGAAAATGGATCTCTC1260    TrpThrGlnLeuTyrLeuIleThrAspTyrHisGluAsnGlySerLeu    305310315    TATGACTTCCTGAAATGTGCTACACTGGACACCAGAGCCCTGCTTAAA1308    TyrAspPheLeuLysCysAlaThrLeuAspThrArgAlaLeuLeuLys    320325330    TTGGCTTATTCAGCTGCCTGTGGTCTGTGCCACCTGCACACAGAAATT1356    LeuAlaTyrSerAlaAlaCysGlyLeuCysHisLeuHisThrGluIle    335340345    TATGGCACCCAAGGAAAGCCCGCAATTGCTCATCGAGACCTAAAGAGC1404    TyrGlyThrGlnGlyLysProAlaIleAlaHisArgAspLeuLysSer    350355360365    AAAAACATCCTCATCAAGAAAAATGGGAGTTGCTGCATTGCTGACCTG1452    LysAsnIleLeuIleLysLysAsnGlySerCysCysIleAlaAspLeu    370375380    GGCCTTGCTGTTAAATTCAACAGTGACACAAATGAAGTTGATGTGCCC1500    GlyLeuAlaValLysPheAsnSerAspThrAsnGluValAspValPro    385390395    TTGAATACCAGGGTGGGCACCAAACGCTACATGGCTCCCGAAGTGCTG1548    LeuAsnThrArgValGlyThrLysArgTyrMetAlaProGluValLeu    400405410    GACGAAAGCCTGAACAAAAACCACTTCCAGCCCTACATCATGGCTGAC1596    AspGluSerLeuAsnLysAsnHisPheGlnProTyrIleMetAlaAsp    415420425    ATCTACAGCTTCGGCCTAATCATTTGGGAGATGGCTCGTCGTTGTATC1644    IleTyrSerPheGlyLeuIleIleTrpGluMetAlaArgArgCysIle    430435440445    ACAGGAGGGATCGTGGAAGAATACCAATTGCCATATTACAACATGGTA1692    ThrGlyGlyIleValGluGluTyrGlnLeuProTyrTyrAsnMetVal    450455460    CCGAGTGATCCGTCATACGAAGATATGCGTGAGGTTGTGTGTGTCAAA1740    ProSerAspProSerTyrGluAspMetArgGluValValCysValLys    465470475    CGTTTGCGGCCAATTGTGTCTAATCGGTGGAACAGTGATGAATGTCTA1788    ArgLeuArgProIleValSerAsnArgTrpAsnSerAspGluCysLeu    480485490    CGAGCAGTTTTGAAGCTAATGTCAGAATGCTGGGCCCACAATCCAGCC1836    ArgAlaValLeuLysLeuMetSerGluCysTrpAlaHisAsnProAla    495500505    TCCAGACTCACAGCATTGAGAATTAAGAAGACGCTTGCCAAGATGGTT1884    SerArgLeuThrAlaLeuArgIleLysLysThrLeuAlaLysMetVal    510515520525    GAATCCCAAGATGTAAAAATCTGATGGTTAAACCATCGGAGGAGAAACTCT1935    GluSerGlnAspValLysIle    530    AGACTGCAAGAACTGTTTTTACCCATGGCATGGGTGGAATTAGAGTGGAATAAGGATGTT1995    AACTTGGTTCTCAGACTCTTTCTTCACTACGTGTTCACAGGCTGCTAATATTAAACCTTT2055    CAGTACTCTTATTAGGATACAAGCTGGGAACTTCTAAACACTTCATTCTTTATATATGGA2115    CAGCTTTATTTTAAATGTGGTTTTTGATGCCTTTTTTTAAGTGGGTTTTTATGAACTGCA2175    TCAAGACTTCAATCCTGATTAGTGTCTCCAGTCAAGCTCTGGGTACTGAATTGCCTGTTC2235    ATAAAACGGTGCTTTCTGTGAAAGCCTTAAGAAGATAAATGAGCGCAGCAGAGATGGAGA2295    AATAGACTTTGCCTTTTACCTGAGACATTCAGTTCGTTTGTATTCTACCTTTGTAAAACA2355    GCCTATAGATGATGATGTGTTTGGGATACTGCTTATTTTATGATAGTTTGTCCTGTGTCC2415    TTAGTGATGTGTGTGTGTCTCCATGCACATGCACGCCGGGATTCCTCTGCTGCCATTTGA2475    ATTAGAAGAAAATAATTTATATGCATGCACAGGAAGATATTGGTGGCCGGTGGTTTTGTG2535    CTTTAAAAATGCAATATCTGACCAAGATTCGCCAATCTCATACAAGCCATTTACTTTGCA2595    AGTGAGATAGCTTCCCCACCAGCTTTATTTTTTAACATGAAAGCTGATGCCAAGGCCAAA2655    AGAAGTTTAAAGCATCTGTAAATTTGGACTGTTTTCCTTCAACCACCATTTTTTTTGTGG2715    TTATTATTTTTGTCACGGAAAGCATCCTCTCCAAAGTTGGAGCTTCTATTGCCATGAACC2775    ATGCTTACAAAGAAAGCACTTCTTATTGAAGTGAATTCCTGCATTTGATAGCAATGTAAG2835    TGCCTATAACCATGTTCTATATTCTTTATTCTCAGTAACTTTTAAAAGGGAAGTTATTTA2895    TATTTTGTGTATAATGTGCTTTATTTGCAAATCACCC2932    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 532 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    MetThrGlnLeuTyrIleTyrIleArgLeuLeuGlyAlaTyrLeuPhe    151015    IleIleSerArgValGlnGlyGlnAsnLeuAspSerMetLeuHisGly    202530    ThrGlyMetLysSerAspSerAspGlnLysLysSerGluAsnGlyVal    354045    ThrLeuAlaProGluAspThrLeuProPheLeuLysCysTyrCysSer    505560    GlyHisCysProAspAspAlaIleAsnAsnThrCysIleThrAsnGly    65707580    HisCysPheAlaIleIleGluGluAspAspGlnGlyGluThrThrLeu    859095    AlaSerGlyCysMetLysTyrGluGlySerAspPheGlnCysLysAsp    100105110    SerProLysAlaGlnLeuArgArgThrIleGluCysCysArgThrAsn    115120125    LeuCysAsnGlnTyrLeuGlnProThrLeuProProValValIleGly    130135140    ProPhePheAspGlySerIleArgTrpLeuValLeuLeuIleSerMet    145150155160    AlaValCysIleIleAlaMetIleIlePheSerSerCysPheCysTyr    165170175    LysHisTyrCysLysSerIleSerSerArgArgArgTyrAsnArgAsp    180185190    LeuGluGlnAspGluAlaPheIleProValGlyGluSerLeuLysAsp    195200205    LeuIleAspGlnSerGlnSerSerGlySerGlySerGlyLeuProLeu    210215220    LeuValGlnArgThrIleAlaLysGlnIleGlnMetValArgGlnVal    225230235240    GlyLysGlyArgTyrGlyGluValTrpMetGlyLysTrpArgGlyGlu    245250255    LysValAlaValLysValPhePheThrThrGluGluAlaSerTrpPhe    260265270    ArgGluThrGluIleTyrGlnThrValLeuMetArgHisGluAsnIle    275280285    LeuGlyPheIleAlaAlaAspIleLysGlyThrGlySerTrpThrGln    290295300    LeuTyrLeuIleThrAspTyrHisGluAsnGlySerLeuTyrAspPhe    305310315320    LeuLysCysAlaThrLeuAspThrArgAlaLeuLeuLysLeuAlaTyr    325330335    SerAlaAlaCysGlyLeuCysHisLeuHisThrGluIleTyrGlyThr    340345350    GlnGlyLysProAlaIleAlaHisArgAspLeuLysSerLysAsnIle    355360365    LeuIleLysLysAsnGlySerCysCysIleAlaAspLeuGlyLeuAla    370375380    ValLysPheAsnSerAspThrAsnGluValAspValProLeuAsnThr    385390395400    ArgValGlyThrLysArgTyrMetAlaProGluValLeuAspGluSer    405410415    LeuAsnLysAsnHisPheGlnProTyrIleMetAlaAspIleTyrSer    420425430    PheGlyLeuIleIleTrpGluMetAlaArgArgCysIleThrGlyGly    435440445    IleValGluGluTyrGlnLeuProTyrTyrAsnMetValProSerAsp    450455460    ProSerTyrGluAspMetArgGluValValCysValLysArgLeuArg    465470475480    ProIleValSerAsnArgTrpAsnSerAspGluCysLeuArgAlaVal    485490495    LeuLysLeuMetSerGluCysTrpAlaHisAsnProAlaSerArgLeu    500505510    ThrAlaLeuArgIleLysLysThrLeuAlaLysMetValGluSerGln    515520525    AspValLysIle    530    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1952 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 187..1692    (D) OTHER INFORMATION: /product="Murine ALK6"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    AAGCGGCGGCAGAAGTTGCCGGCGTGGTGCTCGTAGTGAGGGCGCGGAGGACCCGGGACC60    TGGGAAGCGGCGGCGGGTTAACTTCGGCTGAATCACAACCATTTGGCGCTGAGCTATGAC120    AAGAGAGCAAACAAAAAGTTAAAGGAGCAACCCGGCCATAAGTGAAGAGAGAAGTTTATT180    GATAACATGCTCTTACGAAGCTCTGGAAAATTAAATGTGGGCACCAAG228    MetLeuLeuArgSerSerGlyLysLeuAsnValGlyThrLys    1510    AAGGAGGATGGAGAGAGTACAGCCCCCACCCCTCGGCCCAAGATCCTA276    LysGluAspGlyGluSerThrAlaProThrProArgProLysIleLeu    15202530    CGTTGTAAATGCCACCACCACTGTCCGGAAGACTCAGTCAACAATATC324    ArgCysLysCysHisHisHisCysProGluAspSerValAsnAsnIle    354045    TGCAGCACAGATGGGTACTGCTTCACGATGATAGAAGAAGATGACTCT372    CysSerThrAspGlyTyrCysPheThrMetIleGluGluAspAspSer    505560    GGAATGCCTGTTGTCACCTCTGGATGTCTAGGACTAGAAGGGTCAGAT420    GlyMetProValValThrSerGlyCysLeuGlyLeuGluGlySerAsp    657075    TTTCAATGTCGTGACACTCCCATTCCTCATCAAAGAAGATCAATTGAA468    PheGlnCysArgAspThrProIleProHisGlnArgArgSerIleGlu    808590    TGCTGCACAGAAAGGAATGAGTGTAATAAAGACCTCCACCCCACTCTG516    CysCysThrGluArgAsnGluCysAsnLysAspLeuHisProThrLeu    95100105110    CCTCCTCTCAAGGACAGAGATTTTGTTGATGGGCCCATACACCACAAG564    ProProLeuLysAspArgAspPheValAspGlyProIleHisHisLys    115120125    GCCTTGCTTATCTCTGTGACTGTCTGTAGTTTACTCTTGGTCCTCATT612    AlaLeuLeuIleSerValThrValCysSerLeuLeuLeuValLeuIle    130135140    ATTTTATTCTGTTACTTCAGGTATAAAAGACAAGAAGCCCGACCTCGG660    IleLeuPheCysTyrPheArgTyrLysArgGlnGluAlaArgProArg    145150155    TACAGCATTGGGCTGGAGCAGGACGAGACATACATTCCTCCTGGAGAG708    TyrSerIleGlyLeuGluGlnAspGluThrTyrIleProProGlyGlu    160165170    TCCCTGAGAGACTTGATCGAGCAGTCTCAGAGCTCGGGAAGTGGATCA756    SerLeuArgAspLeuIleGluGlnSerGlnSerSerGlySerGlySer    175180185190    GGCCTCCCTCTGCTGGTCCAAAGGACAATAGCTAAGCAAATTCAGATG804    GlyLeuProLeuLeuValGlnArgThrIleAlaLysGlnIleGlnMet    195200205    GTGAAGCAGATTGGAAAAGGCCGCTATGGCGAGGTGTGGATGGGAAAG852    ValLysGlnIleGlyLysGlyArgTyrGlyGluValTrpMetGlyLys    210215220    TGGCGTGGAGAAAAGGTGGCTGTGAAAGTGTTCTTCACCACGGAGGAA900    TrpArgGlyGluLysValAlaValLysValPhePheThrThrGluGlu    225230235    GCCAGCTGGTTCCGAGAGACTGAGATATATCAGACGGTCCTGATGCGG948    AlaSerTrpPheArgGluThrGluIleTyrGlnThrValLeuMetArg    240245250    CATGAGAATATTCTGGGGTTCATTGCTGCAGATATCAAAGGGACTGGG996    HisGluAsnIleLeuGlyPheIleAlaAlaAspIleLysGlyThrGly    255260265270    TCCTGGACTCAGTTGTACCTCATCACAGACTATCATGAAAACGGCTCC1044    SerTrpThrGlnLeuTyrLeuIleThrAspTyrHisGluAsnGlySer    275280285    CTTTATGACTATCTGAAATCCACCACCTTAGACGCAAAGTCCATGCTG1092    LeuTyrAspTyrLeuLysSerThrThrLeuAspAlaLysSerMetLeu    290295300    AAGCTAGCCTACTCCTCTGTCAGCGGCCTATGCCATTTACACACGGAA1140    LysLeuAlaTyrSerSerValSerGlyLeuCysHisLeuHisThrGlu    305310315    ATCTTTAGCACTCAAGGCAAGCCAGCAATCGCCCATCGAGACTTGAAA1188    IlePheSerThrGlnGlyLysProAlaIleAlaHisArgAspLeuLys    320325330    AGTAAAAACATCCTGGTGAAGAAAAATGGAACTTGCTGCATAGCAGAC1236    SerLysAsnIleLeuValLysLysAsnGlyThrCysCysIleAlaAsp    335340345350    CTGGGCTTGGCTGTCAAGTTCATTAGTGACACAAATGAGGTTGACATC1284    LeuGlyLeuAlaValLysPheIleSerAspThrAsnGluValAspIle    355360365    CCACCCAACACCCGGGTTGGCACCAAGCGCTATATGCCTCCAGAAGTG1332    ProProAsnThrArgValGlyThrLysArgTyrMetProProGluVal    370375380    CTGGACGAGAGCTTGAATAGAAACCATTTCCAGTCCTACATTATGGCT1380    LeuAspGluSerLeuAsnArgAsnHisPheGlnSerTyrIleMetAla    385390395    GACATGTACAGCTTTGGACTCATCCTCTGGGAGATTGCAAGGAGATGT1428    AspMetTyrSerPheGlyLeuIleLeuTrpGluIleAlaArgArgCys    400405410    GTTTCTGGAGGTATAGTGGAAGAATACCAGCTTCCCTATCACGACCTG1476    ValSerGlyGlyIleValGluGluTyrGlnLeuProTyrHisAspLeu    415420425430    GTGCCCAGTGACCCTTCTTATGAGGACATGAGAGAAATTGTGTGCATG1524    ValProSerAspProSerTyrGluAspMetArgGluIleValCysMet    435440445    AAGAAGTTACGGCCTTCATTCCCCAATCGATGGAGCAGTGATGAGTGT1572    LysLysLeuArgProSerPheProAsnArgTrpSerSerAspGluCys    450455460    CTCAGGCAGATGGGGAAGCTTATGACAGAGTGCTGGGCGCAGAATCCT1620    LeuArgGlnMetGlyLysLeuMetThrGluCysTrpAlaGlnAsnPro    465470475    GCCTCCAGGCTGACGGCCCTGAGAGTTAAGAAAACCCTTGCCAAAATG1668    AlaSerArgLeuThrAlaLeuArgValLysLysThrLeuAlaLysMet    480485490    TCAGAGTCCCAGGACATTAAACTCTGACGTCAGATACTTGTGGACAGAGCAAGA1722    SerGluSerGlnAspIleLysLeu    495500    ATTTCACAGAAGCATCGTTAGCCCAAGCCTTGAACGTTAGCCTACTGCCCAGTGAGTTCA1782    GACTTTCCTGGAAGAGAGCACGGTGGGCAGACACAGAGGAACCCAGAAACACGGATTCAT1842    CATGGCTTTCTGAGGAGGAGAAACTGTTTGGGTAACTTGTTCAAGATATGATGCATGTTG1902    CTTTCTAAGAAAGCCCTGTATTTTGAATTACCATTTTTTTATAAAAAAAA1952    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 502 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    MetLeuLeuArgSerSerGlyLysLeuAsnValGlyThrLysLysGlu    151015    AspGlyGluSerThrAlaProThrProArgProLysIleLeuArgCys    202530    LysCysHisHisHisCysProGluAspSerValAsnAsnIleCysSer    354045    ThrAspGlyTyrCysPheThrMetIleGluGluAspAspSerGlyMet    505560    ProValValThrSerGlyCysLeuGlyLeuGluGlySerAspPheGln    65707580    CysArgAspThrProIleProHisGlnArgArgSerIleGluCysCys    859095    ThrGluArgAsnGluCysAsnLysAspLeuHisProThrLeuProPro    100105110    LeuLysAspArgAspPheValAspGlyProIleHisHisLysAlaLeu    115120125    LeuIleSerValThrValCysSerLeuLeuLeuValLeuIleIleLeu    130135140    PheCysTyrPheArgTyrLysArgGlnGluAlaArgProArgTyrSer    145150155160    IleGlyLeuGluGlnAspGluThrTyrIleProProGlyGluSerLeu    165170175    ArgAspLeuIleGluGlnSerGlnSerSerGlySerGlySerGlyLeu    180185190    ProLeuLeuValGlnArgThrIleAlaLysGlnIleGlnMetValLys    195200205    GlnIleGlyLysGlyArgTyrGlyGluValTrpMetGlyLysTrpArg    210215220    GlyGluLysValAlaValLysValPhePheThrThrGluGluAlaSer    225230235240    TrpPheArgGluThrGluIleTyrGlnThrValLeuMetArgHisGlu    245250255    AsnIleLeuGlyPheIleAlaAlaAspIleLysGlyThrGlySerTrp    260265270    ThrGlnLeuTyrLeuIleThrAspTyrHisGluAsnGlySerLeuTyr    275280285    AspTyrLeuLysSerThrThrLeuAspAlaLysSerMetLeuLysLeu    290295300    AlaTyrSerSerValSerGlyLeuCysHisLeuHisThrGluIlePhe    305310315320    SerThrGlnGlyLysProAlaIleAlaHisArgAspLeuLysSerLys    325330335    AsnIleLeuValLysLysAsnGlyThrCysCysIleAlaAspLeuGly    340345350    LeuAlaValLysPheIleSerAspThrAsnGluValAspIleProPro    355360365    AsnThrArgValGlyThrLysArgTyrMetProProGluValLeuAsp    370375380    GluSerLeuAsnArgAsnHisPheGlnSerTyrIleMetAlaAspMet    385390395400    TyrSerPheGlyLeuIleLeuTrpGluIleAlaArgArgCysValSer    405410415    GlyGlyIleValGluGluTyrGlnLeuProTyrHisAspLeuValPro    420425430    SerAspProSerTyrGluAspMetArgGluIleValCysMetLysLys    435440445    LeuArgProSerPheProAsnArgTrpSerSerAspGluCysLeuArg    450455460    GlnMetGlyLysLeuMetThrGluCysTrpAlaGlnAsnProAlaSer    465470475480    ArgLeuThrAlaLeuArgValLysLysThrLeuAlaLysMetSerGlu    485490495    SerGlnAspIleLysLeu    500    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1822 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 49..1341    (C) IDENTIFICATION METHOD: experimental    (D) OTHER INFORMATION: /function="OSTEOGENIC PROTEIN"    /product= "OP1"    /evidence= EXPERIMENTAL    /standard.sub.-- name= "OP1"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GGTGCGGGCCCGGAGCCCGGAGCCCGGGTAGCGCGTAGAGCCGGCGCGATGCACGTG57    MetHisVal    1    CGCTCACTGCGAGCTGCGGCGCCGCACAGCTTCGTGGCGCTCTGGGCA105    ArgSerLeuArgAlaAlaAlaProHisSerPheValAlaLeuTrpAla    51015    CCCCTGTTCCTGCTGCGCTCCGCCCTGGCCGACTTCAGCCTGGACAAC153    ProLeuPheLeuLeuArgSerAlaLeuAlaAspPheSerLeuAspAsn    20253035    GAGGTGCACTCGAGCTTCATCCACCGGCGCCTCCGCAGCCAGGAGCGG201    GluValHisSerSerPheIleHisArgArgLeuArgSerGlnGluArg    404550    CGGGAGATGCAGCGCGAGATCCTCTCCATTTTGGGCTTGCCCCACCGC249    ArgGluMetGlnArgGluIleLeuSerIleLeuGlyLeuProHisArg    556065    CCGCGCCCGCACCTCCAGGGCAAGCACAACTCGGCACCCATGTTCATG297    ProArgProHisLeuGlnGlyLysHisAsnSerAlaProMetPheMet    707580    CTGGACCTGTACAACGCCATGGCGGTGGAGGAGGGCGGCGGGCCCGGC345    LeuAspLeuTyrAsnAlaMetAlaValGluGluGlyGlyGlyProGly    859095    GGCCAGGGCTTCTCCTACCCCTACAAGGCCGTCTTCAGTACCCAGGGC393    GlyGlnGlyPheSerTyrProTyrLysAlaValPheSerThrGlnGly    100105110115    CCCCCTCTGGCCAGCCTGCAAGATAGCCATTTCCTCACCGACGCCGAC441    ProProLeuAlaSerLeuGlnAspSerHisPheLeuThrAspAlaAsp    120125130    ATGGTCATGAGCTTCGTCAACCTCGTGGAACATGACAAGGAATTCTTC489    MetValMetSerPheValAsnLeuValGluHisAspLysGluPhePhe    135140145    CACCCACGCTACCACCATCGAGAGTTCCGGTTTGATCTTTCCAAGATC537    HisProArgTyrHisHisArgGluPheArgPheAspLeuSerLysIle    150155160    CCAGAAGGGGAAGCTGTCACGGCAGCCGAATTCCGGATCTACAAGGAC585    ProGluGlyGluAlaValThrAlaAlaGluPheArgIleTyrLysAsp    165170175    TACATCCGGGAACGCTTCGACAATGAGACGTTCCGGATCAGCGTTTAT633    TyrIleArgGluArgPheAspAsnGluThrPheArgIleSerValTyr    180185190195    CAGGTGCTCCAGGAGCACTTGGGCAGGGAATCGGATCTCTTCCTGCTC681    GlnValLeuGlnGluHisLeuGlyArgGluSerAspLeuPheLeuLeu    200205210    GACAGCCGTACCCTCTGGGCCTCGGAGGAGGGCTGGCTGGTGTTTGAC729    AspSerArgThrLeuTrpAlaSerGluGluGlyTrpLeuValPheAsp    215220225    ATCACAGCCACCAGCAACCACTGGGTGGTCAATCCGCGGCACAACCTG777    IleThrAlaThrSerAsnHisTrpValValAsnProArgHisAsnLeu    230235240    GGCCTGCAGCTCTCGGTGGAGACGCTGGATGGGCAGAGCATCAACCCC825    GlyLeuGlnLeuSerValGluThrLeuAspGlyGlnSerIleAsnPro    245250255    AAGTTGGCGGGCCTGATTGGGCGGCACGGGCCCCAGAACAAGCAGCCC873    LysLeuAlaGlyLeuIleGlyArgHisGlyProGlnAsnLysGlnPro    260265270275    TTCATGGTGGCTTTCTTCAAGGCCACGGAGGTCCACTTCCGCAGCATC921    PheMetValAlaPhePheLysAlaThrGluValHisPheArgSerIle    280285290    CGGTCCACGGGGAGCAAACAGCGCAGCCAGAACCGCTCCAAGACGCCC969    ArgSerThrGlySerLysGlnArgSerGlnAsnArgSerLysThrPro    295300305    AAGAACCAGGAAGCCCTGCGGATGGCCAACGTGGCAGAGAACAGCAGC1017    LysAsnGlnGluAlaLeuArgMetAlaAsnValAlaGluAsnSerSer    310315320    AGCGACCAGAGGCAGGCCTGTAAGAAGCACGAGCTGTATGTCAGCTTC1065    SerAspGlnArgGlnAlaCysLysLysHisGluLeuTyrValSerPhe    325330335    CGAGACCTGGGCTGGCAGGACTGGATCATCGCGCCTGAAGGCTACGCC1113    ArgAspLeuGlyTrpGlnAspTrpIleIleAlaProGluGlyTyrAla    340345350355    GCCTACTACTGTGAGGGGGAGTGTGCCTTCCCTCTGAACTCCTACATG1161    AlaTyrTyrCysGluGlyGluCysAlaPheProLeuAsnSerTyrMet    360365370    AACGCCACCAACCACGCCATCGTGCAGACGCTGGTCCACTTCATCAAC1209    AsnAlaThrAsnHisAlaIleValGlnThrLeuValHisPheIleAsn    375380385    CCGGAAACGGTGCCCAAGCCCTGCTGTGCGCCCACGCAGCTCAATGCC1257    ProGluThrValProLysProCysCysAlaProThrGlnLeuAsnAla    390395400    ATCTCCGTCCTCTACTTCGATGACAGCTCCAACGTCATCCTGAAGAAA1305    IleSerValLeuTyrPheAspAspSerSerAsnValIleLeuLysLys    405410415    TACAGAAACATGGTGGTCCGGGCCTGTGGCTGCCACTAGCTCCTCC1351    TyrArgAsnMetValValArgAlaCysGlyCysHis    420425430    GAGAATTCAGACCCTTTGGGGCCAAGTTTTTCTGGATCCTCCATTGCTCGCCTTGGCCAG1411    GAACCAGCAGACCAACTGCCTTTTGTGAGACCTTCCCCTCCCTATCCCCAACTTTAAAGG1471    TGTGAGAGTATTAGGAAACATGAGCAGCATATGGCTTTTGATCAGTTTTTCAGTGGCAGC1531    ATCCAATGAACAAGATCCTACAAGCTGTGCAGGCAAAACCTAGCAGGAAAAAAAAACAAC1591    GCATAAAGAAAAATGGCCGGGCCAGGTCATTGGCTGGGAAGTCTCAGCCATGCACGGACT1651    CGTTTCCAGAGGTAATTATGAGCGCCTACCAGCCAGGCCACCCAGCCGTGGGAGGAAGGG1711    GGCGTGGCAAGGGGTGGGCACATTGGTGTCTGTGCGAAAGGAAAATTGACCCGGAAGTTC1771    CTGTAATAAATGTCACAATAAAACGAATGAATGAAAAAAAAAAAAAAAAAA1822    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 431 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    MetHisValArgSerLeuArgAlaAlaAlaProHisSerPheValAla    151015    LeuTrpAlaProLeuPheLeuLeuArgSerAlaLeuAlaAspPheSer    202530    LeuAspAsnGluValHisSerSerPheIleHisArgArgLeuArgSer    354045    GlnGluArgArgGluMetGlnArgGluIleLeuSerIleLeuGlyLeu    505560    ProHisArgProArgProHisLeuGlnGlyLysHisAsnSerAlaPro    65707580    MetPheMetLeuAspLeuTyrAsnAlaMetAlaValGluGluGlyGly    859095    GlyProGlyGlyGlnGlyPheSerTyrProTyrLysAlaValPheSer    100105110    ThrGlnGlyProProLeuAlaSerLeuGlnAspSerHisPheLeuThr    115120125    AspAlaAspMetValMetSerPheValAsnLeuValGluHisAspLys    130135140    GluPhePheHisProArgTyrHisHisArgGluPheArgPheAspLeu    145150155160    SerLysIleProGluGlyGluAlaValThrAlaAlaGluPheArgIle    165170175    TyrLysAspTyrIleArgGluArgPheAspAsnGluThrPheArgIle    180185190    SerValTyrGlnValLeuGlnGluHisLeuGlyArgGluSerAspLeu    195200205    PheLeuLeuAspSerArgThrLeuTrpAlaSerGluGluGlyTrpLeu    210215220    ValPheAspIleThrAlaThrSerAsnHisTrpValValAsnProArg    225230235240    HisAsnLeuGlyLeuGlnLeuSerValGluThrLeuAspGlyGlnSer    245250255    IleAsnProLysLeuAlaGlyLeuIleGlyArgHisGlyProGlnAsn    260265270    LysGlnProPheMetValAlaPhePheLysAlaThrGluValHisPhe    275280285    ArgSerIleArgSerThrGlySerLysGlnArgSerGlnAsnArgSer    290295300    LysThrProLysAsnGlnGluAlaLeuArgMetAlaAsnValAlaGlu    305310315320    AsnSerSerSerAspGlnArgGlnAlaCysLysLysHisGluLeuTyr    325330335    ValSerPheArgAspLeuGlyTrpGlnAspTrpIleIleAlaProGlu    340345350    GlyTyrAlaAlaTyrTyrCysGluGlyGluCysAlaPheProLeuAsn    355360365    SerTyrMetAsnAlaThrAsnHisAlaIleValGlnThrLeuValHis    370375380    PheIleAsnProGluThrValProLysProCysCysAlaProThrGln    385390395400    LeuAsnAlaIleSerValLeuTyrPheAspAspSerSerAsnValIle    405410415    LeuLysLysTyrArgAsnMetValValArgAlaCysGlyCysHis    420425430    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 102 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (ix) FEATURE:    (A) NAME/KEY: Protein    (B) LOCATION: 1..102    (D) OTHER INFORMATION: /label=OPX    /note= "Each Xaa is independently selected from a group    of one or more specified amino acids as defined in the    specification"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    CysXaaXaaHisGluLeuTyrValXaaPheXaaAspLeuGlyTrpXaa    151015    AspTrpXaaIleAlaProXaaGlyTyrXaaAlaTyrTyrCysGluGly    202530    GluCysXaaPheProLeuXaaSerXaaMetAsnAlaThrAsnHisAla    354045    IleXaaGlnXaaLeuValHisXaaXaaXaaProXaaXaaValProLys    505560    XaaCysCysAlaProThrXaaLeuXaaAlaXaaSerValLeuTyrXaa    65707580    AspXaaSerXaaAsnValXaaLeuXaaLysXaaArgAsnMetValVal    859095    XaaAlaCysGlyCysHis    100    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    GCGGATCCTGTTGTGAAGGNAATATGTG28    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    GCGATCCGTCGCAGTCAAAATTTT24    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    GCGGATCCGCGATATATTAAAAGCAA26    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    CGGAATTCTGGTGCCATATA20    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    GlyXaaGlyXaaXaaGly    15    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    HisArgAspLeuLysSerLysAsn    15    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    GlyThrLysArgTyrMetAlaProGlu    15    __________________________________________________________________________

What is claimed is:
 1. A method for antagonizing OP-1 binding to a cellsurface receptor, comprising the step of exposing an OP-1 responsivecell, in the presence of OP-1, to a protein selected from the groupconsisting of:(i) a polypeptide comprising amino acids 16-123 of SEQ IDNo:3 (ALK-2); (ii) a polypeptide comprising amino acids 24-152 of SEQ IDNo: 5 (ALK-3); and (iii) a polypeptide comprising amino acids 23-122 ofSEQ ID No: 7 (ALK-6);wherein said polypeptide is provided in an amounteffective for antagonizing the binding of OP-1 to said cell surfacereceptor.
 2. A method for antagonizing OP-1 binding to a cell surfacereceptor, comprisingexposing an OP-1 responsive cell, in the presence ofOP-1, to a polypeptide having binding affinity for OP-1, and having atleast 40% amino acid sequence homology with amino acids 23-122 of SEQ IDNO: 7 (ALK-6);wherein said polypeptide is provided in an amounteffective to antagonize the binding of OP-1 to said cell surfacereceptor.
 3. The method of claim 2, wherein said polypeptide is encodedby a nucleic acid which hybridizes with nucleotides 256-552 of SEQ IDNO: 7.